Novel mixtures for assaying nucleic acid, novel method of assaying nucleic acid with the use of the same and nucleic acid probe to be used therefor

ABSTRACT

[Problems] To provide a novel mixture for assaying a target nucleic acid, characterized by enabling a nucleic acid assay while: 1) requiring no step of diluting the target nucleic acid; 2) requiring no procedure of changing a probe concentration depending on a concentration of the target nucleic acid. [Means for Solving Problems] 1) A mixture which comprises one internal standard nucleic acid and two nucleic acid probes labeled with a fluorescent dye; 2) a mixture for measuring Km value which comprises one internal standard nucleic acid having a partial mutation and one nucleic acid probe labeled with a fluorescent dye; 3) a mixture which comprises one internal standard nucleic acid and one double nucleic acid probe labeled with two fluorescent dyes; and a method for assaying a nucleic acid by making use thereof.

TECHNICAL FIELD

This invention relates to a novel mixture for assaying a nucleic acid,and specifically to a novel mixture for assaying accurately,conveniently and non-expensively one or plural nucleic acids, and amethod for assaying a nucleic acid using the same, and nucleic acidprobes to be used for the assaying method and a nucleic acid-assayingkit.

BACKGROUND ART

Many examples of methods for assaying a target nucleic acid are methodsfor assaying a nucleic acid in a homogenous solution by using afluorescent dye-labeled target nucleic acid probe having propertieschanging in fluorescence on hybridizing with a target nucleic acid(called a nucleic acid probe for a homogenous solution system or atarget nucleic acid probe, having the same meaning). The method does notrequire, by this characteristics, 1) any step immobilizing a targetnucleic acid (or any step washing a probe) indispensable for commonhybridizing method and 2) any step washing out non-reacting probes (orany step immobilizing non-reacting genes); the method, therefore, is amethod for assaying easily, rapidly and accurately a target nucleicacid. With such reasons, the method for assaying quantitatively a targetnucleic acid has been in a wide used for various methods for analyzinggenes (see non-patent reference 1).

In assaying directly a target nucleic acid by a nucleic acid probe for ahomogenous solution system, an object could be achieved by using thefollowing procedures.

(1) making a target nucleic acid of known concentrations readybeforehand hybridize with a nucleic acid probe for a homogenous solutionsystem, and, on this hybridization, monitoring a change or the amount ofchange in an optical character;

(2) preparing a calculating curve for determining a target nucleic acidby preparing an equation relating to the above change or amount ofchange in an optical character and amounts of a target nucleic acidbecause the change or the amount of change is positively proportional toamounts of a target nucleic acid;

(3) conducting a procedures similar to the above procedures in regard toan unknown sample, and determining the amount of a target nucleic acidfrom the above calculating curve based on the obtained change or amountof change in an optical character.

In the method, however, if a target nucleic acid exists greater inconcentration than an added nucleic acid probe, a change or the amountof a change in an optical character is at any time stationary. Becauseof this, the conventional methods need any of the means, 1) diluting atarget nucleic acid sample, and 2) preparing in advance plural assayingsystems with a probe having various concentrations. The above 1)requires a diluting processing step; the operation become complicated.As results, the above 2) has such problems that (1) long assaying timeis needed; (2) diluting errors occur; and (3) on automation of theassaying, an for dilution is needed. In the above 2) also, the reactiontime and reaction temperature suitable for hybridization vary withresponse to the concentrations of an added probe (if a target nucleicacid and a target nucleic acid probe are higher in concentrations, thetime for completed hybridization becomes shorter; if contrary to theformer, the reaction time becomes longer. If a target nucleic acid and aprobe are higher in concentrations, a Tm value is higher; if contrary tothe former, it becomes lower); as a result, there is such problems that(1) an assaying system needs to be optimized in every concentration of aprobe, and (2) a calculating curve needs to be prepared in everyconcentration of an added probe.

Non-patent reference 1: Protein, Nucleic Acid and Enzyme; vol. 35, No.17, Kyoritu Shuppan Co., Ltd.; Experimental Medical; vol. 15, No. 7,1997, Yodosha Co., Ltd.

DISCLOSURE OF THE INVENTION

Problems to be Solved by Invention

With the foregoing circumstances in view, the present invention has asan object to provide a novel mixture for assaying a target nucleic acid,characterized by enabling a nucleic acid assaying method to assay atarget nucleic acid without requiring any processing step of diluting ofa target nucleic acid and any procedures of changing a probeconcentration, a novel method for assaying a nucleic acid using thesame, and a novel nucleic acid probe to be used for these.

MEANS TO BE SOLVED PROBLEMS

As a result of an extensive investigation, the present inventors haveobtained the following findings and completed the present invention.That is, a target nucleic acid is added to a reaction solution (ahybridizing reaction system) comprising a known concentration of aspecific internal standard nucleic acid, a specific target nucleic acidprobe labeled with a fluorescent dye for a homogenous solution systemand/or a specific internal standard nucleic acid probe labeled with afluorescent dye and making it possible to hybridize the above internalstandard nucleic acid, and then, is allowed to make a hybridizingreaction; while an occurred change of an optical character is measured.Further a measured-value ratio of the internal standard nucleic acid andthe target nucleic acid is determined; these steps enabled the presentinvention to be completed.

Described specifically, the present invention provides:

1) Novel mixture or novel reaction solution (hereinafter, the bothtogether are collectively called simply a “novel mixture”.) for assayingone or two or more target nucleic acids, which comprises one or two ormore below nucleic acid probes for a homogenous solution system and oneor two or more below internal standard nucleic acid, or further one ortwo or more below internal standard nucleic acid probe:

A) Nucleic acid probe for homogenous solution system (hereinafter,called a “target nucleic acid probe”) having below characteristics:

a) said target nucleic acid probe is formed of one strandedoligonucleotide;

b) said target nucleic acid probe is labeled with one or two or moremolecule of fluorescent dyes of one or two or more kinds at least one ofboth end portions and/or at least one of base portions in the chain, atleast one of sugar moieties and/or at least one of phosphate moieties ofthe oligonucleotide;

c) said target nucleic acid probe enables a fluorescent character tochange on hybridizing with a target nucleic acid and/or an internalstandard nucleic acid;

d) said target nucleic acid probe is capable of hybridizing withoutdiscriminating with a target nucleic acid or an internal standardnucleic acid;

e) said target nucleic acid probe is capable of producing a differencebetween the amount of a change in a fluorescent character on hybridizingwith an internal standard nucleic acid and that on hybridizing with atarget nucleic acid;

B) Internal standard nucleic acid (called an “internal gene” or an“internal standard gene” in a case): wherein said internal standardnucleic acid has a structure different in at least a portion from thestructure of a target nucleic acid of a region corresponding to theabove target nucleic acid probe, and is capable of producing adifference between the amount of a change in a fluorescent characterproduced on hybridizing with the above target nucleic acid probe and oneon the hybridization of a target nucleic acid with the above targetnucleic acid probe;

C) Internal standard nucleic acid probe:

wherein said internal standard nucleic acid probe has the followingcharacteristics:

said internal standard nucleic acid probe has the above characteristicsa) to e) of the above target nucleic acid probe, wherein a fluorescentlabeling portion and a fluorescent character of a labeled fluorescentdye each are different from those of the above target nucleic acidprobe;

2) a novel mixture according to the above 1), wherein said targetnucleic acid probe is a probe having a base sequence not complementaryto a target nucleic acid in a partial region;

3) a novel mixture according to the above 1), wherein if said nucleicacids are labeled with two or more fluorescent dyes, the probes arelabeled with the fluorescent dyes which are with each other different intheir optical character;

4) a novel mixture according to the above 1), wherein the amount of achange in an optical character is the increased amount (for example, aprobe labeled with dyes each causing an FRET phenomena) or the decreasedamount (for example, a Q probe);

5) a novel mixture according to the above 1) or 3), wherein said targetnucleic acid probe and/or said internal standard nucleic acid probeare/is a probe labeled at least two portions (end portions, baseportions in an chain, sugar moieties, phosphate moieties) withfluorescent dyes with different fluorescent character occurred onhybridization of a target nucleic acid probe and/or an internal standardnucleic acid probe with a target nucleic acid and/or an internalstandard nucleic acid (hereinafter, these probes are called simply “adoubly-labeled nucleic acid probe”);

6) a novel mixture according to the above 5), wherein said at least twoportions are at least two bases;

7) a novel mixture according to the above 6), wherein said bases arecytosine (hereinafter a cytosine is called simply a “C”);

8) a novel mixture according to the above 7, wherein C's are bases ofboth ends;

9) a novel mixture according to the above 1) or 5), wherein a basesequence of a target nucleic acid probe is complementary to a targetnucleic acid and an internal standard nucleic acid excluding both baseend portions (portions comprising from the 1^(st) base to the 3^(rd)base in length; the end base being counted as the “1^(st) base”);

10) a novel mixture according to the above 5), wherein saiddoubly-labeled nucleic acid probe is a doubly-labeled nucleic acid probemaking at one portion of the labeled portions a difference between theamount of a change in a fluorescent character on hybridization with atarget nucleic acid and that on hybridization with an internal standardnucleic acid, but not making such a difference at the other portion;

11) a novel mixture according to the above 1) or 5), wherein a basesequence of said target nucleic acid probe or said doubly-labelednucleic acid is at least complementary to a target nucleic acid or aninternal standard nucleic acid excluding both end base portions (a basesequence of from at least the 1^(st) base to the 3^(rd) base in length;with the end base being counted as the 1^(st) base);

12) a novel mixture according to the above 1) or 5), wherein a basesequence of said target nucleic acid probe or said doubly-labelednucleic acid probe is not complementary to a target nucleic acid and/oran internal standard nucleic acid at an end portion opposite an endportion labeled with a fluorescent dye;

13) a novel mixture according to the above 1) or 5), wherein said targetnucleic acid or said doubly-labeled nucleic acid has at least a basesequence completely complementary to a target nucleic acid and aninternal standard nucleic acid;

14) a novel mixture according to the above 1) or 5), wherein, if thebase of a target nucleic acid corresponding to an end base of a targetnucleic acid probe or a doubly-labeled nucleic acid probe is counted as1^(st) base, the number of a G of the corresponding target nucleic acidand internal standard nucleic acid in a end base sequence of from the1^(st) base to the 3^(rd) base is larger in a target nucleic acid thanin an internal standard nucleic acid, or smaller in a target nucleicacid than in an internal standard nucleic acid;

15) a novel mixture according to the above 1) or 5), wherein, if thebase of a target nucleic acid corresponding to both end base of a targetnucleic acid probe or a doubly-labeled nucleic acid probe is counted asthe 1^(st) base, the number of G of the corresponding target nucleicacid and internal standard nucleic acid in a end base sequence of fromthe 1^(st) base to the 3^(rd) base is larger in a target nucleic acidthan in an internal standard nucleic acid in one end region, and in theother end region smaller in a target nucleic acid than or equal in aninternal standard nucleic acid in other end region; or in one end regionsmaller in a target nucleic acid than in an internal standard nucleicacid and in the other end region larger in a target nucleic acid than orequal in an internal standard nucleic acid;

16) a novel mixture according to the above 1) or 5), wherein the base ofa target nucleic acid corresponding to one end base of a target nucleicacid probe or a doubly-labeled nucleic acid probe is a base other than aG and the base corresponding to the other end base is a G;

17) a novel mixture according to the above 1) or 5), wherein if any oftwo bases of a target nucleic acid corresponding to both end bases of atarget nucleic acid probe is a G, that of an internal standard nucleicacid is a base other than a G; and if that of a target nucleic acid is abase other than a G, that of an internal standard nucleic acid is a G;

18) a novel mixture according to the above 1) or 5), wherein, if thebase of a target nucleic acid corresponding to both end base of a targetnucleic acid probe or a doubly-labeled nucleic acid probe is counted asthe 1^(st) base, the corresponding base sequence of a target nucleicacid and internal standard nucleic acid in a end base sequence of fromthe 1^(st) base to the 3^(rd) base is different in one end region, butthe same in the other end region;

19) a novel mixture according to the above 1) or 5), wherein saidmixture comprises further one or two or more sorts of D)fluorescence-quenching substance enabling the fluorescence of a targetnucleic acid probe or a doubly-labeled nucleic acid probe having nothybridized with a target nucleic acid and an internal standard nucleicacid to reduce, or one or two or more sorts of oligonucleotide labeledwith E) the fluorescence-quenching substance (hereinafter, called simply“quenching substance-labeled probe”);

20) a novel mixture according to the above 19), wherein said quenchingsubstance-labeled probe has the below characteristics (1) and/or (2):

The characteristics:

(1) The dissociating temperature of the hybrid complex of a targetnucleic acid probe or a doubly-labeled nucleic acid probe and afluorescent quenching substance-labeled probe is lower than thedissociating temperature of the hybrid complex of a target nucleic acidprobe or a doubly-labeled nucleic acid probe and a nucleic acid or aninternal standard nucleic acid.

(2) After the hybridization of a target nucleic acid probe or adoubly-labeled nucleic acid probe with a target nucleic acid, or afterthe hybridization of a target nucleic acid probe or a doubly-labelednucleic acid probe with a target nucleic acid, a fluorescent quenchingsubstance-labeled probe can hybridize with a probe for a homogenoussolution system or a doubly-labeled nucleic acid probe due to thecharacteristics of the above (1).

21) a novel mixture according the above 1), wherein said novel mixturecomprises a) an internal standard nucleic acid, b) a target nucleic acidprobe and/or c) an internal standard nucleic acid probe according to theabove 1), and d) exonuclease under the below conditions or exonucleaseattached to the novel mixture as a kit;

the conditions: a target nucleic acid probe and/or an internal standardnucleic acid probe are not complementary to at least one of a targetnucleic acid and an internal standard nucleic acid at afluorescent-labeled portion region (the region: 1 (one) to 3 base inlength, preferably 1 (one) base);

22) a novel mixture according to the above 21), wherein said exonucleaseis 3′→5′exonuclease, 5′→3′ exonuclease, S1 nuclease or Mung BeanNuclease;

23) a novel mixture according to the above 1), wherein said novelmixture comprises one or two or more pairs of internal standard probesand target nucleic acid probes;

24) a novel method for assaying a target nucleic acid, comprisingassaying one or two or more target nucleic acids using the novel mixtureaccording to the above 1);

25) a novel method for assaying a target nucleic acid according to theabove 24), wherein said novel method comprises:

conducting a hybridizing reaction under the presence of one or two ormore target nucleic acids and/or one or two or more internal standardnucleic acids;

measuring a change in a fluorescent character of the reaction system,which change is derived from a fluorescent dye labeled at a targetnucleic acid probe and/or an internal standard nucleic acid probe andcalculating a ratio of the obtained measuring values; and

determining concentrations of one or two or more target nucleic acidsbased on the obtained ratio;

26) a novel method for assaying one or two or more target nucleic acidsaccording to the above 24), wherein said target nucleic acid is anucleic acid amplified by a gene amplification method (until any phaseof an initial phase, a middle phase and a stationary phase);

27) a novel method for assaying one or two or more target nucleic acidsaccording to the above 24), wherein said target nucleic acid is at leastone of a target nucleic acid and internal standard nucleic acidamplified by a gene amplification method;

28) a novel method for assaying one or two or more target nucleic acidsaccording to the above 24), wherein said target nucleic acid is at leastone of a target nucleic acid and internal standard nucleic acidamplified by a gene amplification method using a same primer set;

29) a novel method for assaying one or two or more target nucleic acidsaccording to the above, wherein said novel mixture according to any oneof the above 1) to 23) comprises the following target nucleic acid probeor doubly-labeled nucleic acid probe and a known concentration of aninternal standard nucleic acid according to any one of the above 1) to20);

Target nucleic acid probe (hereinafter, this kind of a probe will becalled a “recognizable nucleic acid probe”): a target nucleic acid probeaccording to any one of the above 1) to 23) is not complementary to anyone of a target nucleic acid and an internal standard nucleic acid atleast any one or two or more ones of portions labeled with a fluorescentdye(s).

Doubly-labeled nucleic acid probe (hereinafter, this sort of a probewill be called a “recognizable doubly-labeled nucleic acid”): adoubly-labeled nucleic acid probe according to any one of the above

5) to 23) is not complementary to any one of a target nucleic acid andan internal standard nucleic acid at any one of portions labeled withany one of two sorts of fluorescent dyes.

30) a novel method for assaying one or two or more target nucleic acidsaccording to the above 29), wherein said novel method comprises:

measuring the amount of changes in fluorescent characters derived from atarget nucleic acid probe and an internal standard nucleic acid probe;

calculating a ratio of the amount of a change in a fluorescent characterfrom the target nucleic acid probe to the amount of a change in afluorescent character from the internal standard nucleic acid probebased on the obtained measuring values;

calculating a constituent ratio of the target nucleic acid and theinternal standard nucleic acid based on the obtained ratio; and

determining a concentration of a target nucleic acid based on theobtained constituent ratio;

31) a novel method for assaying one or two or more target nucleic acids,wherein, in the method for assaying a nucleic acid according to theabove 24) or 29), the fluorescence emission are made to quench for atarget nucleic acid probe or internal standard nucleic acid probe nothybridized with any of an internal standard nucleic acid and a targetnucleic acid;

32) a novel method for assaying one or two or mote target nucleic acidsaccording to the above 31), wherein said quenching of a fluorescentemission is conducted by using a quenching substance having an effectquenching a fluorescence emission and/or a quenching substance-labeledprobe having the same effect;

33) a calculating equation represented by the following equation forcalculating accurately a target nucleic acid based on measuring valuesof a change in an optical character in the novel method for assaying atarget nucleic acid according to the above 24) or 29);x=(−a′−B+Ba′+b′+A−Ab′)/(b′−b−Ab′+Ab−a′+a+Ba′−Ba)wherein said equation is valid under the below conditions; and saidsigns have the below meanings:

The conditions are as follows: in the novel method, the doubly-labelednucleic acid probe is used, wherein said nucleic acid probe is labeledwith dyes A and B.

The signs are as follows:

y: a proportion of a hybridized probe;

1−y: a proportion of non-hybridized probe;

x: a proportion of a target gene;

1−x: a proportion of an internal standard gene;

A: a ratio of the fluorescent intensity of dye A of the doubly-labelednucleic acid probe on no hybridization in use of a practical sample tothe fluorescent intensity of dye A of the doubly-labeled nucleic probeon no hybridization;

a: a ratio of the fluorescent intensity of dye A of the doubly-labelednucleic acid probe on its 100%-hybridization with a target nucleic acidto the fluorescent intensity of dye A of the doubly-labeled nucleic acidprobe on no hybridization;

a′: a ratio of the fluorescent intensity of dye A of the doubly-labelednucleic acid probe on its 100%-hybridization with an internal standardnucleic acid to the fluorescent intensity of dye A of the doubly-labelednucleic acid probe on no hybridization;

B: a ratio of the fluorescent intensity of dye B of the doubly-labelednucleic acid probe on no hybridization using a practical sample to thefluorescent intensity of dye B of the doubly-labeled nucleic probe on nohybridization;

b: a ratio of the fluorescent intensity of dye B of the doubly-labelednucleic acid probe on its 100%-hybridization with a target gene to thefluorescent intensity of dye B of the doubly-labeled nucleic acid probeon no hybridization;

b′: a ratio of the fluorescent intensity of dye B of the doubly-labelednucleic acid probe on its 100%-hybridization with an internal standardnucleic acid to the fluorescent intensity of dye B of the doubly-labelednucleic acid probe on no hybridization.

34) a calculating equation represented by the following equation forcalculating accurately a target nucleic acid based on measuring valuesof a change in an optical character in the novel method for assaying atarget nucleic acid according to the above 24) or 29);x=(b−B−Ab+A+a′B−a′)/(a′B−a′−aB+a)wherein said equation is valid under the below conditions; and saidsigns have the below meanings:

The conditions are as follows: in the novel method, the doubly-labelednucleic acid probe is used, wherein said nucleic acid probe is labeledwith dyes A and B.

The signs are as follows:

y: a proportion of a hybridized probe;

1−y: a proportion of non-hybridized probe;

x: a proportion of a target gene;

1−x: a proportion of an internal standard gene;

A: a ratio of the fluorescent intensity for dye A of the doubly-labelednucleic acid probe on use of a practical sample to the fluorescentintensity for dye A of non-hybridizing doubly-labeled nucleic probe;

a: a ratio of the fluorescent intensity for dye A of the doubly-labelednucleic acid probe on its 100%-hybridization with a target nucleic acidto the fluorescent intensity for dye A of non-hybridizing doubly-labelednucleic acid probe;

a′: a ratio of the fluorescent intensity for dye A of the doubly-labelednucleic acid probe on the 100%-hybridization of the doubly-labelednucleic acid probe with an internal standard nucleic acid to thefluorescent intensity for dye A of the doubly-labeled probe on100%-hybridization of the doubly-labeled nucleic acid probe with thequenching substance-labeled probe;

B: a ratio of the fluorescent intensity of dye B of the doubly-labelednucleic acid probe on use of a practical sample to the fluorescentintensity of dye B of the doubly-labeled nucleic probe on its100%-hybridization with a quenching substance-labeled nucleic acidprobe;

b: a ratio of the fluorescent intensity of dye B of the doubly-labelednucleic acid probe on its 100%-hybridization with a target gene and aninternal standard gene to the fluorescent intensity of dye B of thedoubly-labeled nucleic acid probe on its 100%-hybridization with aquenching substance-labeled nucleic acid probe.

35) A kit for assaying a nucleic acid, comprising a novel mixtureaccording to the above 1) or 5);

36) a novel method for assaying a nucleic acid, which comprises:amplifying one or plural target nucleic acids and an internal standardnucleic acid in a reaction system comprising one or plural internalstandard nucleic acids until an optional phase of from a beginning phaseto a stationary phase by a gene-amplification method; and determiningstarting concentrations prior to the amplification of one or pluraltarget nucleic acids by using the resultant reaction or the amplifiedproduct as a sample;

37) a method for assaying one or plural nucleic acids prior to anamplification according to the above 32), wherein saidgene-amplification method is a method for amplifying a target nucleicacid and an internal standard nucleic acid by using a primer set commonto the amplification of the target nucleic acid and to that of aninternal standard nucleic acid;

38) a method for assaying one or plural nucleic acids prior toamplification according to the above 36), wherein the primer for saidamplification is a Q probe;

39) a novel mixture for assaying one or plural target nucleic acidsbased on measurement of a Tm value, which comprises a pair of a belownucleic acid and a below internal standard nucleic acid;

The target nucleic acid probe: which is one-strand oligonucleotidelabeled with one or two or more fluorescent dyes, wherein saidoligonucleotide is capable of hybridizing with a target nucleic acid andan internal standard nucleic acid, and enables a change of a fluorescentcharacter of the labeled fluorescent dyes on hybridization with thenucleic acid and internal standard nucleic acid, wherein in pluralnucleic acid probes the fluorescent dyes of the probes each aredifferent.

The internal standard nucleic acid: in which the base sequence of aportion of said internal standard nucleic acid hybridizing with saidnucleic acid probe is different in part from the base-sequence of aportion of a target nucleic acid hybridizing with said nucleic acidprobe.

40) a novel mixture for assaying one or plural target nucleic acidsbased on measurement of a Tm value according to the above 39), whereinsaid nucleic acid probe is a single stranded oligonucleotide labeledwith a fluorescent dye at a cytosine portion of the oligonucleotide;

41) a novel mixture for assaying one or plural target nucleic acidsbased on measurement of a Tm value according to the above 39), whereinsaid change in a fluorescent character for a fluorescent dye is adecrease in fluorescent intensity;

42) a novel mixture for assaying one or plural target nucleic acidsbased on measurement of a Tm value according to the above 35), whereinsaid change in a fluorescent character for a fluorescent dye is anincrease in fluorescent intensity;

43) a novel method for assaying a target nucleic acid, which comprisesmeasuring fluorescent intensity using said novel mixture according tothe above 39) with changing temperature under the presence of pluraltarget nucleic acids; and determining a target nucleic acid by thefollowing procedures:

the procedures comprising:

1) drawing a curve responding to a change in fluorescent intensitymeasured;

2) differentiating the resulting curve;

3) integrating the resulting peak(s) and determining the area(s) of thepeak(s);

4) calculating a ratio(s) between the resulting peak area (s) of theinternal standard nucleic acid and the resulting peak area(s) of thetarget nucleic acid;

5) multiplying the concentration of the internal standard nucleic acidby said ratio.

44) a novel method for assaying a nucleic acid according to the above37), wherein said target nucleic acid is a nucleic acid productamplified by a gene amplification method in a reaction solution systemcontaining an internal standard nucleic acid according to the above 35);

45) a novel method for assaying a nucleic acid according to the above43), wherein said primer is a Q probe;

46) a kit for assaying a nucleic acid, which comprises a novel mixtureaccording to the above 1) or 39);

47) a target nucleic acid probe or a doubly-labeled nucleic acid probe,in which said target nucleic acid probe or doubly-labeled nucleic acidprobe is described above and has at least any one of the belowstructures:

1. structures of said target nucleic acid probe, wherein

(1) said structure has a portion not complementary to a target nucleicacid and/or an internal standard nucleic acid at a end portion or bothend portions;

(2) in the above (1), said nucleic acid probe is labeled with afluorescent dye at one portion not complementary to the target nucleicacid and/or an internal standard nucleic acid, having a cytosine (a C)or a guanine (a G) in a range of 1 to 3 bases from the labeled base in afluorescent dye-labeled portion (the labeled base is numbered as 1(one));

(3) in the above (1), the one other portion not complementary to atarget nucleic acid and/or an internal standard nucleic acid is aportion opposite the one end portion labeled with a fluorescent dye;

(4) in the above (1), one other portion not complementary to a targetnucleic acid and/or an internal standard nucleic acid is in a range of 1(one) to 4 bases as the number of bases;

(5) in the above (1), if any of two bases of a target nucleic acidcorresponding to the both ends of the target nucleic acid probes is a G,that of the internal standard nucleic acid is a base other than a G; ifthat of the target nucleic acid is a base other than a G, that of aninternal standard is a G.

2. Structures of a doubly-labeled target nucleic acid probe, wherein,

(6) portions labeled with fluorescent dyes were at least two bases;

(7) the bases according to the above (6) are two C's;

(8) the two C's according to the above (7) are the bases of both ends;

(9) the base sequence according to the above (6) are complementary to atarget nucleic acid or an internal standard nucleic acid excluding bothend portion (at least from the 1^(st) base to the 3^(rd) base, the endbase being counted as the 1^(st) base);

(10) the doubly-labeled nucleic acid probe according to the above (6) or(9) is a doubly-labeled nucleic acid probe making at one portion adifference between the amount of change in a fluorescent character onhybridization with a target nucleic acid and that on hybridization withan internal standard nucleic acid, but not making such a difference atthe other portion;

3. Structures common to a target nucleic acid probe and a doubly-labelednucleic acid probe: wherein

(11) in any one of the above (1) to (10), a base sequence of a targetnucleic acid probe or a doubly-labeled nucleic acid is at leastcomplementary to a target nucleic acid or an internal standard nucleicacid excluding to both end base portions (a base sequence of at leastfrom the 1^(st) base to the 3^(rd) base; the end base being counted asthe 1^(st) base);

(12) in any one of the above (1) to (11), a target nucleic acid probe ora doubly-labeled nucleic acid probe has a base sequence completelycomplementary to a target nucleic acid and an internal standard nucleicacid;

(13) in any one of the above (1) to (12), if the base of a targetnucleic acid corresponding to an end base of a target nucleic acid probeor a doubly-labeled nucleic acid probe is taken as the 1^(st) base, thenumber of a G of the corresponding target nucleic acid and internalstandard nucleic acid in a end base sequence of from the 1^(st) base tothe 3^(rd) base is larger in a target nucleic acid than in an internalstandard nucleic acid, or smaller in a target nucleic acid than in aninternal standard nucleic acid;

(14) in any one of the above (1) to (13), if the base of a targetnucleic acid corresponding to both end base of a target nucleic acidprobe or a doubly-labeled nucleic acid probe is taken as the 1^(st)base, the number of G of the corresponding target nucleic acid andinternal standard nucleic acid in a end base sequence of from the 1^(st)base to the 3^(rd) base is larger in a target nucleic acid than in aninternal standard nucleic acid in one end region, and in other endregion smaller in a target nucleic acid than or equal in an internalstandard nucleic acid in other end rejoin; or in one end region smallerin a target nucleic acid than in an internal standard nucleic acid andin other end region larger in a target nucleic acid than or equal in aninternal standard nucleic acid;

(15) a novel mixture according to the above 1) or 5), wherein, in anyone of the above (1) to (14), the base of a target nucleic acidcorresponding to one end base of a target nucleic acid probe or adoubly-labeled nucleic acid probe is a base other than a G and the basecorresponding to the other end base is a G.

(16) in any one of the above (1) to (15), if any of two bases of atarget nucleic acid corresponding to both end bases a target nucleicacid probe is a G, that of an internal standard nucleic acid is otherthan a G; and if that of a target nucleic acid is other than a G, thatof an internal standard nucleic acid is a G;

(17) in any one of the above, if the base of a target nucleic acidcorresponding to both end base of a target nucleic acid probe or adoubly-labeled nucleic acid probe is taken as the 1^(st) base, thecorresponding base sequence of a target nucleic acid and internalstandard nucleic acid in a end base sequence of from the 1^(st) base tothe 3^(rd) base is different in the one end region, but the same in theother end region.

ADVANTAGEOUS EFFECT OF THE INVENTION

A novel method having the following characteristics is completed byusing the above novel mixture according to the present invention. Thatis, in the method for assaying a nucleic acid, 1) processing steps fordiluting a target nucleic acid are not required; 2) a change of theconcentration of a nucleic acid for use with response to theconcentration of a target nucleic acid are not required; 3) pluraltarget nucleic acids can be assayed in a like assaying system; 4) anassaying sensitivity is enhanced; and 5) the combination of a method forassaying a nucleic acid according to the present invention and a methodfor amplifying a nucleic acid provides the following merits:

(1) subsequent to the completion of a gene amplifying reaction, thecombined method makes it possible to assay in a rapid and convenient waya nucleic acid without opening a reaction tube for the gene amplifyingreaction; therefore, it does not require a PCR post processing step andcan simply, easily and rapidly assay a nucleic acid; (2) to open agene-amplifying reaction tube is not needed; the combined method doesnot have any risk of contamination with amplified products; (3) theassay is hard to be affected by inhibitors because the combined methodis a competitive method; (4) since a gene-amplifying processing-step anda detecting processing step of a amplified product can be completelydivided, a large quantity-sample becomes to be treated; and asample-treating power can be conveniently and non-expensively improved(for example, subsequent to the gene-amplification by using pluralnon-expensive PCR-apparatuses not having any fluorescence-measuringfunction, by analyzing obtained data in order using afluorescence-measuring apparatus, using even one fluorescence-measuringapparatus, a large quantitative-sample can be treated.); (5) a nucleicacid becomes to be assayed by a markedly simple and non-expensivemeasuring apparatus because the amplifying processing of a nucleic acidis needed not to be monitored in a real time way and the measuringapparatus is needed to have a thermally-cycling function indispensableto measuring a PCR processing.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates an outline of a method for detecting a target geneand an internal standard gene by using a Qprobe.

FIG. 2 illustrates an outline of a method for assaying quantitatively agene by a dissociation curve(Tm) analysis using a Qprobe.

FIG. 3 illustrates an outline of a method for assaying quantitatively agene by using a doubly-labeled Qprobe (a switching probe).

FIG. 4 illustrates an outline of the principle for a competitive PCRmethod.

FIG. 5 illustrates an outline of the relation of afluorescence-quenching ratio and a constituent ratio of genes.

◯ total gene amount=200 nM, ▪ total gene amount=800 nM

FIG. 6 illustrates the relation of a fluorescence-quenching ratio and aconstituent ratio of genes (in using a switching probe).

FIG. 7 illustrates the relation of a fluorescence quenching ratio and aconstituent ratio of genes prior to a nucleic acid-amplificationreaction (PCR) (in using an end-point gene-quantitatively-assayingmethod through a gene-amplification method using a switching probe).

FIG. 8 illustrates the experimental procedures in Example 2

FIG. 9 illustrates an outline of the method for assaying quantitativelya constituent ratio of genes by using a target nucleic acid probe.

FIG. 10 illustrates the relation of a constituent ratio of genes and afluorescence-quenching ratio in the use of a probe for a homogenoussolution system.

□ total gene-amount: 200 nM; ◯ total gene-amount: 400 nM; ♦ totalgene-amount: 800 nM

FIG. 11 illustrates a outline of the method for detecting a gene using adoubly-labeled nucleic acid probe (its part 2)

FIG. 12 illustrates the relation between a ratio of afluorescence-quenching ratio in BODIPY FL dye to afluorescence-quenching ratio in TAMURA dye and a constituent ratio ofgenes of a target gene/an internal standard gene.

◯ gene amount: 200 nM; ♦ gene amount: 400 nM; Δ total gene amount: 800nM

FIG. 13 illustrates an outline of a method for detecting a gene by usinga doubly-labeled nucleic acid probe (its part 3).

FIG. 14 illustrates a difference between a calculating curve withcorrection in TAMURA dye and a calculating curve with no correction inTAMURA dye.

● total gene-amount: 200 nm; ▴ total gene-amount: 800 nM

FIG. 15 is an illustrative figure concerning conditions of a probe and afluorescent change.

FIG. 16 illustrates an relative equation as to a proportion of a targetgene and a fluorescence-quenching ratio

FIG. 17 illustrates the detection of a target gene and an internalstandard gene under the no presence of a quenching substance-labelednucleic acid probe or under the presence of it.

FIG. 18 illustrates an outline of a method for using an enzyme having a3′→5′exonuclease activity.

FIG. 19 illustrates a relation of a fluorescence-measuring-value ratioand a constituent ratio of genes.

▪ 100 nM; ⋄ 800 nM

FIG. 20 illustrates an outline of a method for assaying quantitatively agene by using a combination of a doubly-labeled nucleic acid probe and aquenching substance-labeled nucleic acid probe.

BEST MODES FOR CARRYING OUT THE INVENTION

The present invention will hereinafter be described in detail. Beforedescribing the present invention in detail, however, definitions will beprovided for certain terms used throughout the application including theclaims. It is to be noted that the terms employed in the presentinvention have the same meanings as those used commonly in molecularbiology, genetics or genetic engineering, or microbiology or microbialengineering unless otherwise specifically indicated.

The terms “one or two or more”, “one or two or mote” and “one or plural”as used herein mean at least one”.

The term “target nucleic acid” as used herein means a nucleic acid thedetection or quantitatively assaying of which is intended.

A gene should be compassed in the terms “nucleic acid”, “target nucleicacid” and “internal standard nucleic acid”. In this specification, asgeneral terms, “a target nucleic acid”, “an internal standard nucleicacid”, or simply “nucleic acid” are used; as specifical terms, “targetgene”, “internal standard gene”, or simply “gene” are used.

The term “a nucleic acid contained in a sample” may be also calledsimply a target nucleic acid or an objective nucleic acid.

The expression “to assay a nucleic acid” or “to measure theconcentration of a nucleic acid” as use herein means to perform aquantitative detection of the nucleic acid, to perform a qualitativedetection of the nucleic acid, to simply measure or simply monitor theintensity of fluorescence from a nucleic acid polymerization system, orto measure a change or the amount of a change in an optical character oftarget nucleic acid(s) in an assaying reaction system by using a pluralwavelength and then to calculate a ratio between the measuring values,followed by to determine the concentration of the target nucleic acidwith the ratio. The above expression should also be interpreted toencompass an operation or the like that the data obtained as describedabove is studied by the known method of Kurata et al. (EP 1 046 717 A9)to determine the concentration (the number of copies or the like) of anucleic acid existing in a single system.

Based on the above reason, the term “a nucleic acid contained in asample” as used herein should be not only limited to any specificnucleic acid(s) to be assayed, but also should be interpreted to includean unspecified nucleic acid capable of be detected by the methodaccording to the present invention with no intention. Needless say, itencompasses genes and the like. These nucleic acids may exist together.In addition, no limitation is imposed on the concentration or size ofthose. These nucleic acids should be interpreted to encompass further aDNA, an RNA and the modified nucleic acids thereof.

The term “optical character” means one of various absorption spectra andfluorescence emission spectrum of a fluorescent dye, quencher or thelike, with which a nucleotide is labeled, or its optical characteristicor the like such as absorption intensity, polarization, fluorescenceemission, fluorescence intensity, fluorescence lifetime, fluorescencepolarization or fluorescence anisotropy (these optical characteristicswill be collectively called “fluorescence intensity”). It may also meana characteristic determined by totally analyzing one or more measurementvalues of at least one fluorescent dye or the like, with which a labelednucleotide or the like is labeled, as measured at least one measurementwavelength. For example, a fluorescence intensity curve or the like of amodification reaction of a nucleic acid can be used as an opticalcharacter.

Further, as a general term, a “optical character” is used; as a specificterm, a “fluorescent intensity”, or simply “fluorescence” is used.

In the present invention, the expression “from a change or the amount ofa change in fluorescence intensity” shall embrace not only a change influorescence intensity on the basis of a nucleic acid polymersynthesized in the present invention, but also a change or the amount ofa change in fluorescence intensity when a nucleic acid probe for ahomogeneous solution system, said nucleic acid probe having been labeledwith a fluorescent dye and/or quencher, is hybridized with the amplifiednucleic acid.

A hybridization complex between a primer probe and a correspondingnucleic acid is called a “hybrid” or a “hybrid complex”, or simply a“nucleic acid-primer complex” or a “primer-nucleic acid complex”.

In the present invention, for example, such terms are used that anucleic acid probe “complements” or “is complementary to”, or inaddition “does not complement” or “is not complementary to” a targetnucleic acid at a partial region of the probe. These terms “complements”or “is complementary to” means that when two kinds of oligonucleotidesexist in a single system, the nucleic acids can bind each other's atcorresponding bases by hydrogen bonding. In addition, it means that oneof the nucleic acids can hybridize with the other. Further, such a termis used that a partial region of a nucleic acid probe “corresponds with”a partial region of a target nucleic acid: the term “correspond with” inthis instance has no concept of the hydrogen bonding betweencorresponding bases; and it means that simply the nucleic acids have arelation of one to one. Accordingly, the term “correspond” means both of“complement” or “be complementary to” and “do not complement” or “be notcomplementary to”.

The term a “change ratio in an optical character” as used the presentinvention means a ratio of an optical measuring-value of a fluorescentdye or the like in a reaction system or an assaying system on nohybridization of a nucleic acid probe (a target nucleic acid probe, aninternal standard nucleic acid probe) with a target nucleic acid and/oran internal standard nucleic acid to that on hybridization. As anexample, an calculating equation such as (a measuring value onhybridization)/(a measuring value on no hybridization)×100 may beillustrated. If the change is a quenching in fluorescence, it is calleda “fluorescence-quenching ratio”; while if an emission in fluorescence,a “fluorescent emission ratio” or the like. The hybridization occurspreferably at 10° C. to 90° C.; the no hybridization occurs at 90° C. ormore. There is an intermediate hybridization; accordingly, it ispreferable to measure accurately every each experiment.

Incidentally, a portion at which a nucleic acid probe according to thepresent invention is labeled with a fluorescent dye according to thepresent invention is called a “fluorescent dye-labeled portion” or“fluorescence-labeled portion”; these terms have, however, the samemeanings.

The term “fluorescent dye (which may also be called ‘fluorescentsubstance’)” as used in the present invention generally means afluorescent dye which is generally used to label a nucleic acid probe toassay or detect the nucleic acid. Illustrative are fluorescein and itsderivatives [for example, fluorescein isothiocyanate (FITC) and itsderivatives], Alexa 488, Alexa 532, cy3, cy5, 6-joe, EDANS, rhodamine 6G(R6G) and its derivatives [for example, tetramethylrhodamine (TMR),tetramethylrhodamine isothiocynate (TMRITC), and x-rhodamine], Texasred, “BODIPY FL” (“BODIPY” is a trademark, “FL” is a tradename; productof Molecular Probes Corporation, U.S.A.; this will hereinafter applyequally), “BODIPY FL/C3”, “BODIPY FL/C6”, “BODIPY 5-FAM”, “BODIPY493/504”, “BODIPY TMR”, and their derivatives (for example, “BODIPY TR”,“BODIPY R6G”), and “BODIPY 564”, “BODIPY 581”, TAMRA, Pacific Blue(Tradename; Molecular Probes Co., USA), and the like.

Among the above-exemplified fluorescent dyes, TAMRA, FITC, EDANS, TexasRed, 6-joe, TMR, Alexa 488, Alexa 532, “BODIPY FL/C3”, “BODIPY R6G”,“BODIPY FL”, “BODIPY FL/C6”, “BODIPY TMR”, 5-FAM, “BODIPY 493/503”,“BODIPY 564”, “BODIPY 581”, Cy3, Cy5, x-Rhodamine, Pacific Blue and thelike can be mentioned as preferred ones.

The term “quencher” means a substance, which acts on the above-describedfluorescent dye and reduces or quenches the emission of fluorescencefrom the fluorescent dye. Illustrative are Dabcyl, “QSY7” (product ofMolecular Probes Corporation), “QSY33” (product of Molecular ProbesCorporation), Ferrocene and its derivatives, methyl viologen, andN,N′-dimethyl-2,9-diazopyrenium, BHQ, Eclipse, with Dabcyl beingpreferred.

By labeling a specific position of an oligonucleotide with a fluorescentsubstance and a quencher substance as above, the fluorescent emission ofthe fluorescent substance is susceptible to a quenching effect of aquencher.

On a method for amplifying nucleic acid as used in the present inventionmay be not imposed any limitation, if it serves the achievement of anobject of the present invention.

A method for amplifying a nucleic acid as used in the present inventionis a method for amplifying in vitro a nucleic acid. It may be a knownone or a unknown one. It should encompass, for example, a PCR method, anLCR method (ligase chain reaction), a TAS method, an ICAN method(Isothermal and Chimeric primer-initiated Amplification of Nucleicacids), an LAMP method, an NASRA method, an RCA method, a TAMA method, aUCAN method and the like. A PCR method using primer probes or a simpleprobe is preferable.

The above PCR method can preferably have any system.

It should encompass, for example, a quantitative PCR method, a real-timequantitative PCR method, an RT-PCR method, an RNA-primed PCR method, anStretch PCR method, a reverse PCR method, an Alu-sequence-using PCRmethod, a multiple PCR method, a mixed-primer probe-using PCR method, aPNA-using PCR method, and also a method for examining or analyzing adissociating curve in regard to a nucleic acid amplified by a PCRmethod.

A “Q probe” or Qprobe” is a probe proposed by KURATA et al. (Kurata etal., Nucleic Acids Research, 2001, Vol. 29, No. 6, e34). This probe is anucleic acid probe for a homogeneous solution system, in which asingle-stranded oligonucleotide is labeled with a fluorescent dye. Thebase labeled with the fluorescent dye is a G or a C, or there is a G ora C at a position 1 to 3 bases apart from a base in a target nucleicacid, said base corresponding to a labeled base and being counted as the1^(st) base.

A labeling portion of an oligonucleotide to be labeled with afluorescent dye can be an end portion or in its chain. A labelingposition thereof can be a sugar moiety, a phosphate moiety or a basemoiety. The labeling can be labeled on the 3′-OH group or 2′-OH group ofthe sugar moiety at the 3′-end, or the 5′-OH group of the sugar moietyobtained by dephosphorylation at the 5′-end. In the phosphate portion,the phosphate group can be replaced with a sulfonate group, or sulfinylgroup. The labeling portion is preferably a portion containing a C or aG or their selves, with a portion containing a C being the mostpreferable.

The present invention comprises four invention, with each comprisingfurther sub-invention.

A novel mixture according to the present invention can be in a liquid, apowder, a tablet, or a capsule; any can be preferably used; and the formis in particular not limited. In the following description, no form ofthe novel mixture is, therefore, mentioned.

In addition, the following target nucleic acid or an internal standardnucleic acid should encompass an amplified product until an optionalamplification phase (including a stationary phase).

Further, a target nucleic acid should contain a product amplifiedconcurrently in a reaction system from together a target nucleic acidand an internal standard nucleic acid corresponding thereto, itsresultant reaction solution, or an one isolated therefrom together withthe target nucleic acid and internal standard nucleic acid. In thiscase, a same probe can be used, or not be used. A nucleicacid-amplification method can be a conventional one as described below.

The nucleic acid probe (a target nucleic acid and/or an internalstandard nucleic acid) as used in the present invention is a nucleicacid probe changing its optical character; a nucleic acid probedecreasing or increasing in a fluorescent intensity can be exemplified.For example, exemplified can be the above mentioned Qprobe as onedecreasing in a fluorescent intensity, a nucleic acid probe labeled withtwo dyes related to FRET phenomena as one increasing (Proc. Natl. Acad.Sci. USA, Vol. pp 8790-8794, 1988; U.S. Pat. No. 4,996,143; JPH05-50152A; JP H08-313529A; JP H10-215897A; JP Application H11-292861)and the like.

A) First Invention

The present invention is related to a novel mixture or novel reactionsolution (hereinafter, the both together are collectively called simplya “novel mixture”.) for assaying one or two or more target nucleicacids, which comprises one or two or more below nucleic acid probes fora homogenous solution system and one or two or more below internalstandard nucleic acid or further below internal standard nucleic acidprobes:

A) A nucleic acid probe for a homogenous solution system (hereinafter,called “the target nucleic acid probe”) having below characteristics:

a) It is formed of a single strand oligonucleotide. It has a length of10 to 100 bases, preferably 15 to 60 bases, more preferably 20 to 40bases. It can be oligodeoxynucleotide or an oligoribonucleotide. It canalso be a chimeric oligonucleotide.

b) one molecule of it is labeled with one or two or more molecules offluorescent dyes and with one or two or more kinds of fluorescent dyesat an end portion and/or a base portion in the chain of theoligonucleotide, and at a sugar moiety and/or a phosphate moiety;

c) it can cause the labeling fluorescent dye to change in an fluorescentcharacter on hybridizing with any one of a target nucleic acid and aninternal standard nucleic acid;

d) it can hybridize with both a target nucleic acid and an internalstandard nucleic acid;

e) it can produce a difference between the amount of a change in afluorescent character before and after on hybridizing with an internalstandard nucleic acid and that on hybridizing with a target nucleicacid;

f) if it is labeled with two kinds or more of fluorescent dyes, it candiscriminate each between a target nucleic acid and an internal standardnucleic acid based on a change in an fluorescent character onhybridization;

g) its chain length is preferably the same as that of an internalstandard nucleic acid or similar to;

h) its chain length is preferably the same as that of an internalstandard nucleic acid probe or similar to.

B) An internal standard nucleic acid:

a) It comprises a single strand oligonucleotide. Its chain length is 40to 2000 bases, preferably 60 to 500 bases, more preferably 80 to 150bases. The oligonucleotide can be an oligodeoxynucleotide or anoligoribonucleotide. Those can be a chimeric oligonucleotide; and

b) it has a structure different in at least a portion from structure ofa target nucleic acid of a region corresponding to the above targetnucleic acid probe, and is capable of producing a difference between theamount of change in a fluorescent character produced before and after onhybridizing with the above target nucleic acid probe and one on thehybridization of a target nucleic acid with the above target nucleicacid probe.

C) Internal standard nucleic acid probe, it having the followingcharacteristics:

It has the above characteristics a) to h) of the above target nucleicacid probe, wherein a fluorescent labeling portion and the fluorescentcharacter of a labeled fluorescent dye each are different from the onesof the above target nucleic acid probe;

The present invention relates to a novel mixture, wherein the abovetarget nucleic acid probe, internal standard nucleic acid, and aninternal standard nucleic acid as included therein each can havepreferably at least any one of the following structures, and each thosecan preferably have the following relationship.

1) Target Nucleic Acid Probe:

(1) It is at least complementary to a target nucleic acid;

(2) it is complementary to an internal nucleic acid except for at leastone or two or more regions;

(3) it has a region wherein the one or two or more region of it are notcomplementary to a target nucleic acid;

(4) if it is labeled with two or more kinds of fluorescent dyes, thestructures of the labeled portions are at least different from eachothers';

(5) the above structure are a base sequence;

(6) the above base sequence is at least 2 or 3 or more bases as theintegral number of a base;

(7) the portions which have complementary structures as mentioned aboveare labeled with fluorescent dyes;

(8) the portions which have non-complementary structures as mentionedabove are labeled with fluorescent dyes;

(9) a fluorescent dye-labeled portion of the above target nucleic acidis a guanine (a G) base portion and/or a cytosine (a C) portion, or aportion wherein a G and/or a C exists in a range near a base of alabeled portion (in a range from the 1st base to the 3rd base to a3′-end or 5′-end (the labeled base being counted as the 1^(st) base)

(10) the portion labeled with a fluorescent dye as mentioned above is a3′- or 5′-end portion;

(11) the end portion as described above is an end base moiety, a sugarmoiety (any one of a 2′- or 3′-OH group of the 3′-end, and a 5′-OH group(being obtainable by dephosphorylation)), and a phosphate moiety (or thephosphate moiety should encompass a sulfonate group or a sulfite group).

(12) the plural probes as described above are different from eachother's;

(13) the nucleic acid probe as described above is labeled at least twoportions (end portions, base moieties in the chain, sugar moieties,phosphate moieties) with fluorescent dyes having characters differentfrom each other's (hereinafter, called “a doubly-labeled nucleic acidprobe”); (13-2) the fluorescent dyes with fluorescent charactersdifferent from each other's are fluorescent dyes not producing FRET(fluorescence resonance energy transfer) at two portions; (13-3) thefluorescent dyes with fluorescent characters different from each other'sare fluorescent dyes producing FRET at two portions;

(14) the at least two portions as described in the above (12) are atleast two bases; the length as the number of a base between two bases is1 (one) to 100 bases, preferably 10 to 60 bases, more preferably 20 to40 bases;

(14-2) two portions with such distance between those that no FRETphenomena occur are labeled with the fluorescent dyes having differentfluorescent character as described above;

(14-3) two portions with such distance between those that FRET phenomenaoccur are labeled with two fluorescent dyes having different fluorescentcharacter as described above;

(15) the base as described in the above (12) is cytosine (hereinafterabbreviated “C”);

(16) C's as described in the above (12) are bases of both ends;

(17) the base sequence of the target nucleic acid probe as describedabove is complementary to a target nucleic acid and an internal standardnucleic acid excluding both base end portions (two portions comprisingfrom the 1st base to the 3rd base in length; the end base is counted asthe 1^(st) base);

(18) the doubly-labeled nucleic acid as described above is adoubly-labeled nucleic acid probe making at one portion a differencebetween the amount of a change in a fluorescent character onhybridization with a target nucleic acid and that on hybridization withan internal standard nucleic acid, but not making such a difference atthe other portion;

(19) The base sequence of said target nucleic acid probe or saiddoubly-labeled nucleic acid as described above is at least complementaryto a target nucleic acid or an internal standard nucleic acid excludingboth end base portions (a base sequence of at least from the 1st to the3rd base in length; the end base is counted as the 1^(st) base);

(20) the base sequence of the target nucleic acid probe or saiddoubly-labeled nucleic acid probe as described above has a base sequencecomplementary to a target nucleic acid and/or an internal standardnucleic acid;

(21) if the base of a target nucleic acid corresponding to an end baseof a target nucleic acid probe or a doubly-labeled nucleic acid probe istaken as the 1^(st) base, the number of G of the corresponding targetnucleic acid and internal standard nucleic acid in a base sequence theend portion of a range of the 1^(st) base to the 3^(rd) base is largerin a target nucleic acid than in an internal standard nucleic acid, orsmaller in a target nucleic acid than in an internal standard nucleicacid;

(22) if the base of a target nucleic acid corresponding to both end baseof a target nucleic acid probe or a doubly-labeled nucleic acid probe isassumed as the 1^(st) base, the number of a G of the correspondingtarget nucleic acid and internal standard nucleic acid in a basesequence of the end portion in a range of from the 1^(st) base to the3^(rd) base is larger in a target nucleic acid than in an internalstandard nucleic acid in one end region, and in the other end regionsmaller in a target nucleic acid than or equal in an internal standardnucleic acid in the other end region; or in one end region smaller in atarget nucleic acid than in an internal standard nucleic acid; and inthe other end region larger in a target nucleic acid than or equal in aninternal standard nucleic acid;

(23) the base of a target nucleic acid corresponding to one end base ofa target nucleic acid probe or a doubly-labeled nucleic acid probe is abase other than G and the base corresponding to the other end base is aG;

(24) if any of two bases of a target nucleic acid corresponding to bothend bases of a target nucleic acid probe is a G, that of an internalstandard nucleic acid is a base other than a G; and if that of a targetnucleic acid is a base other than a G, that of an internal standardnucleic acid is a G;

(25) a novel mixture according to the above 1) or 5), wherein, if thebase of a target nucleic acid corresponding to both end bases of atarget nucleic acid probe or a doubly-labeled nucleic acid probe isassumed as 1 (one), the corresponding base sequence of a target nucleicacid and internal standard nucleic acid in a end base sequence of fromthe 1st base to the 3rd base is different in one end region, but thesame in the other end region;

(26) the nucleic acid probe and doubly-labeled nucleic acid probe asdescribed above are a fluorescence-quenching probe (for example, aQprobe);

(27) the nucleic acid probe and doubly-labeled nucleic acid probe asdescribed above are a fluorescence-emitting probe;

(27) the doubly-labeled nucleic acid probe as described above is labeledwith a quenching dye and a fluorescence-emitting dye;

(28) when the target nucleic acid probe or doubly-labeled target nucleicacid probe as described above hybridizes with a target nucleic acid oran internal standard nucleic acid, the above target nucleic acid probehas a base sequence designed so that at least one G is present in arange of from the 1^(st) base to 3^(rd) base to the 3′end- or5′end-direction from the labeled base (the labeled base is counted asthe 1^(st) base);

(29) the end portion opposite the fluorescent dye-labeling portion asdescribed above is not complementary to a target nucleic acid and/or aninternal standard nucleic acid (an optional base sequence of from the1st base to the 5th base, with a preferable base sequence being of fromthe 1st base to the 3rd base, wherein an end base is counted in theabove number of the base sequence.);

(30) at least one of the both ends as described above can be any of aoligonucleotide, trinucleotide and mononucleotide, wherein thesenucleotides may be formed of a deoxynucleotide or riboxynucleotide.

(31) the position of the fluorescent dye-labeling as described above inthe end sugar moiety is any one of the OH group of the 5° C. of the5′-end sugar (the OH group being formed by dephosphorylation) and the OHgroup of the 3° C. or 2° C. of the 3′-end sugar (the 2′-OH group is inthe case of a ribonucleotide);

(32) in the presence of at least one of G and C in two regions of atarget nucleic acid, the target nucleic acid probe or doubly-labelednucleic acid probe as described above is a probe having a basesequence(s) designed so that the base sequence(s) is corresponding toone or two of these regions including a G or a C (corresponding to theabove regions), wherein the base sequence is labeled with a fluorescentdye.

A novel mixture according to the present invention comprising, inaddition, one or two or more fluorescence-quenching substances capableof quenching the fluorescence of a target nucleic acid probe or adoubly-labeled nucleic acid probe not hybridizing with a target nucleicacid or an internal standard nucleic acid, or one or two or moreoligonucleotides labeled with the above substance (hereinafter, called a“quenching substance-labeled probe) in addition to the components of themixture as described above.

The novel mixture as described above, wherein the above quenchingsubstance-labeled probe has the following characteristics (1) and/or(2):

Characteristics:

(1) The dissociating temperature of the hybrid complex of a targetnucleic acid probe or a doubly-labeled nucleic acid probe and afluorescent quenching substance-labeled probe is lower than thedissociating temperature of the hybrid complex of a target nucleic acidprobe or a doubly-labeled nucleic acid probe and a nucleic acid or aninternal standard nucleic acid.

(2) After the hybridization of a target nucleic acid probe or adoubly-labeled nucleic acid probe with a target nucleic acid, and afterthe hybridization of a target nucleic acid probe or a doubly-labelednucleic acid probe with a target nucleic acid, a fluorescent quenchingsubstance-labeled probe can hybridize with a probe for a homogenoussolution system or a doubly-labeled nucleic acid probe due to the abovecharacteristics (1).

2) Internal Standard Nucleic Acid

it has at least one of the following characteristics:

(1) An internal standard nucleic acid is not complementary to a targetnucleic acid in one or two or more regions.

(2) An internal standard nucleic acid is not complementary to a targetnucleic acid in one or two or more regions.

(3) An internal standard nucleic acid id complementary to a targetnucleic acid in all regions.

(4) There is G or C in a portion region of the internal standard nucleicacid corresponding to a fluorescence-labeled portion of a target nucleicacid probe.

(5) There is not a G or a C in a portion region of the internal standardnucleic acid corresponding to a fluorescence-labeled portion of a targetnucleic acid probe.

(6) There is not a G or a C in a portion region of the internal standardnucleic acid corresponding to one or two or more fluorescence-labeledportions of a target nucleic acid probe.

(7) There is a G or a C in the portion regions of the internal standardnucleic acid corresponding to one of two or more fluorescence-labeledportions of the target nucleic acid probe; but there is not a G or a Cin the portion regions corresponding to another fluorescence-labeledportions of the target nucleic acid probe.

(8) There is a G or a C in a portion region of the internal standardnucleic acid corresponding to any one of fluorescence-labeled portionsof a doubly-labeled nucleic acid probe.

3) Relation Between Target Nucleic Acid and Internal Standard NucleicAcid.

(1) In such case that a target nucleic acid probe is complementary to atarget nucleic acid at the fluorescent dye-labeled region of the targetnucleic acid probe, the target nucleic acid probe is not complementaryto an internal standard nucleic acid at the region.

(2) In such case that a target nucleic acid probe is complementary to aninternal standard nucleic acid at the fluorescent dye-labeled region ofthe target nucleic acid probe, the target nucleic acid probe is notcomplementary to a target nucleic acid at the region.

4) Internal Standard Nucleic Acid Probe

The internal standard nucleic acid probe has at least the samecharacteristics as that of the above target nucleic acid probe and thesame structure, but its fluorescent dye-labeled portion has a structuralportion different from that of the above target nucleic acid probe. Forexample, the fluorescent dye-labeled portion of the above target nucleicacid probe is the 3′end; while that of the internal standard nucleicacid is the 5′end. In contrary to the former, further its relationbecome contrary in response to this.

B. Second Invention

It is a novel method for assaying a nucleic acid, characterized byassaying one or two or more target nucleic acids by using the abovenovel mixture according to the first invention. No limitation is imposedon this invention. All a method for assaying a nucleic acid by using theabove mixture according to the first invention is within the scope ofthe present invent.

A target nucleic acid is an optional nucleic acid and in particular notlimited. For example, the method can have various modes by thecombination of the above probe for a homogenous solution system and theinternal standard nucleic acid, and further by the combination of thoseand the internal standard nucleic acid probe. For example,

1) It is a reaction product amplified by a gene-amplifying method (aproduct amplified until an optional phase reaching to a stationary phase(any phase of an initial phase, a middle phase and a stationary phase)).

2) It is at least one of a target nucleic acid and an internal standardnucleic acid amplified by a gene amplifying method.

3) It is at least one of a target nucleic acid and internal standardnucleic acid amplified by using a same primer.

The method can have various modes by the combination of the above probefor a homogenous solution system and the internal standard nucleic acid,and further by the combination of those and the internal standardnucleic acid probe. For example, the second invention according to thepresent invention is divided largely to the following modes.

Incidentally, a specific method for assaying a nucleic acid according tothe present invention comprises, any instance, conducting ahybridization in the presence of a target nucleic acid and/or aninternal standard nucleic acid; measuring a change in an opticalcharacter of a reaction system, with the change being derived from afluorescent dye of a target nucleic acid probe and/or an internalstandard nucleic acid probe; calculating a ratio of the obtainedmeasuring values or a ratio of measured changing ratios of the opticalcharacter; and determining a concentration of the target nucleic acidbased on the calculated ratios.

Assaying Method A:

The method comprises conducting a hybridization in the presence of oneor two or more target nucleic acids and/or internal standard nucleicacids, and determining concentrations of one or plural target nucleicacids.

Assaying Method B:

The method comprises assaying a target nucleic acid by using a nucleicacid probe capable of discriminating a target nucleic acid and aninternal standard nucleic acid according to the present invention (anucleic acid capable of discriminating).

Assaying Method C:

In the above method, this method comprises assaying a target nucleicacid by quenching the fluorescence from a target nucleic acid probe orinternal standard nucleic acid probe not hybridizing with an internalstandard nucleic acid and a target nucleic acid.

Assaying Method D:

In the above method, this method comprises adding, subsequent to thehybridization, an exonuclease into an assaying system, and measuring achange in an optical character of an assaying system.

The assaying method A will first described.

Assaying Method A:

This method is divided by combining a probe for a homogenous solutionsystem with an internal standard probe, and further divided by combiningthe combined method with an internal standard nucleic acid probe in thefollowing way.

(1) A method using a mixture comprising the above probe for a homogenoussolution system and an internal standard nucleic acid.

This method can be divided by a method for measuring an assaying systemto the following two methods.

(2) In the above (1), this method is a method using a doubly-labelednucleic acid in stead of the above probe for a homogenous solutionsystem.

(3) In the above (1), this method is a method using a mixture comprisingan internal standard nucleic acid probe.

Next, the above methods will be described in the above order byillustrating a specific target nucleic acid probe, internal standardnucleic acid, or further an specific internal standard nucleic acidprobe. More specifically, these methods will be illustrated in theexamples. The examples should not limit the present invention.

Assaying Method 1-1 (see Example 5 and FIG. 9)

Preferable examples will be next described.

FIG. 9 illustrates a method for assaying a nucleic acid according thepresent invention, in which the simplest labeled nucleic acid andinternal standard nucleic acid are used.

The illustrated method adopts a Q probe (called a “Qprobe”) as a targetnucleic acid probe. Any of the above probes is preferably usable as atarget nucleic acid probe in the present invention. The most preferabletarget nucleic acid probe is a probe in which an end portion opposite afluorescent dye-labeled portion is not complementary to a target nucleicacid and/or an internal standard nucleic acid (an optional base sequencecomprising a range of from the 1st base to the 5th base, preferably of 1to 3 bases; in this counting an end base is counted as the 1st base).

The simplest target nucleic acid and internal standard nucleic acid areillustrated in FIG. 1, in which a singly-labeled nucleic acid isillustrated as a Qprobe.

Target Nucleic Acid Probe:

In addition, the target nucleic acid probe, the singly-labeled probe, isdesigned so that a change (decreasing (quenching)) in an opticalcharacter (fluorescent emission) of a labeling fluorescent dye occurs onits hybridization with a target nucleic acid. That is, a probe, as asingly-labeled nucleic acid probe, is arranged such that, as a basesequence of the portion of the target nucleic acid capable ofhybridizing with the singly-labeled nucleic acid probe, the basecorresponding to the fluorescence-labeled end base of thissingly-labeled nucleic acid probe is a G or a C; and a basecorresponding to non-fluorescence-labeled end base is a base other thana G. On other hand, a base sequence is chosen as a base sequence of theportion of an internal standard nucleic acid capable of hybridizing withthe singly-labeled nucleic acid probe such that the base correspondingto the fluorescence-labeled end base of this singly-labeled nucleic acidprobe is a base other than a G; and a base corresponding tonon-fluorescence-labeled end base is a G or a C. In this case, the basesequence of the singly-labeled nucleic acid probe should be a basesequence complementary to a target nucleic acid and an internal standardnucleic acid except for bases of both ends. The base of afluorescence-labeled end of the singly-labeled nucleic acid probe shouldbe complementary to the target nucleic acid, but not to the internalstandard nucleic acid; the base of a non-fluorescence-labeled end shouldbe contrary to the fluorescence-labeled end. These are in detailindicated in FIG. 9A.

In addition, the singly-labeled nucleic acid probe is possible to designso that, when it hybridizes with an internal standard nucleic acid, achange (decreasing (quenching) in an optical character of a fluorescentdye used for the singly-labeled nucleic acid probe occurs). A basesequence in the region of the internal standard nucleic acid hybridizingwith the singly-labeled nucleic acid probe should be arranged so that abase of the internal standard nucleic acid corresponding to afluorescence-labeled end base of the singly-labeled nucleic acid probeis a G or a C and a base corresponding to a fluorescence-unlabeled endbase is a base other than a G. On the other hand, the base sequence inthe region of the target nucleic acid hybridizing with thesingly-labeled nucleic acid probe should be chosen so that a base of theinternal standard nucleic acid corresponding to the fluorescence-labeledend base of the singly-labeled nucleic acid probe is a base other than aG, and a base corresponding to a non-fluorescence-labeled end base is aG or a C. In this instance, a base sequence of the singly-labelednucleic acid probe should be a base sequence complementary to theinternal standard nucleic acid and the target nucleic acid withexcluding both end bases; the fluorescence-labeled end base of thesingly-labeled nucleic acid probe should be complementary to theinternal standard nucleic acid, but not complementary to the targetnucleic acid. In the non-fluorescence-labeled end base, the abovematters should be contrary. FIG. 9B demonstrates the above description.

Internal Standard Nucleic Acid

A base sequence of the internal standard nucleic acid should preferablybe identical to the target nucleic acid except both end bases. In thefollowing description, a preferable internal standard nucleic acid isdescribed.

The base of the internal standard nucleic acid corresponding to the baseof a fluorescent dye-labeled portion of the above probe should bedesigned so as to be complementary to the base of a fluorescentdye-labeled portion of the above probe. That is, when the base of afluorescent dye-labeled portion of the above probe is a C, the base ofthe internal standard nucleic acid corresponding to that of the aboveprobe should be a G; or, when the base of a fluorescent dye-labeledportion of the above probe is a G, the base of the internal standardnucleic acid corresponding to that of the probe should be a C. Inaddition, the base of the internal standard nucleic acid correspondingto the base of a fluorescent dye-unlabeled portion of the probe shouldbe designed so as to be not complementary to the base of the probe. Itsbase is preferably an adenine (an A) or a thymine (a T).

On the other hand, the base sequence region of a target nucleic acidshould be chosen such that, when the base of a fluorescent dye-labeledportion of the probe is a C, the base of a target nucleic acidcorresponding to that base is a G; or when the base of a fluorescentdye-unlabeled portion of the probe is a G, the base of the targetnucleic acid corresponding to that base is a C; and the base of thetarget nucleic acid corresponding to the base of a fluorescentdye-labeled portion of the probe is not complementary to the base of thefluorescent dye-labeled portion of the probe.

On the basis of the above description, the number of base-pairs formedbetween the target nucleic acid probe and a target nucleic acid and GCcontents of the probe or the nucleic acid become equivalent to thatbetween the target nucleic acid probe and the internal standard nucleicacid and GC contents of the nucleic acid or the internal standardnucleic acid. As a result, there is almost no difference between theheat stability of the hybridizing complex formed by the target nucleicacid probe and the target nucleic acid and that by the internal standardnucleic acid probe and the target nucleic acid. Owing to those, the bothprobes is considered to hybridize equally with the target nucleic acidand the internal standard nucleic acid.

The above described pattern is related to a pattern such thatfluorescence increases on the hybridization of a singly-labeled nucleicacid probe with an internal standard nucleic acid; while the methodhaving a pattern contrary to the above pattern (namely, a patternshowing decreasing fluorescence upon the hybridization of thesingly-labeled nucleic acid with the target nucleic acid) is practicablein the present invention. In other wards, the base sequence of theinternal standard nucleic acid should be designed such that, when thebase of a fluorescent dye-labeled portion of the singly-labeled nucleicacid probe is a C, the base of the internal standard nucleic acidcorresponding to that of the probe is a G; or when the base of afluorescent dye-unlabeled portion of the probe is a G, the base of theinternal standard nucleic acid corresponding to that of the fluorescentdye-unlabeled portion of the probe is a C; and the base of the internalstandard nucleic acid corresponding to the base of a fluorescentdye-labeled portion of the probe is not complementary to that of theprobe. The pattern is suitably shown in FIG. 8A.

Further, these patterns will be more practically interpreted under usingFIG. 9. In FIG. 9A, a Qprobe receives a markedly fluorescence-quenchingeffect (decreased intensity of fluorescence; hereinafter having the sameconcept) on hybridization with a target nucleic acid; such effectproduces from the presence of a G in a position complementary tofluorescence-labeled (fluorescent dye-labeled) a C. The base sequence ofan internal standard gene should be designed so that an adenine (an A)of the gene presents in a position complementary to a C of the 5′-end ofa Qprobe; and a G of the gene presents in a position complementary to aC of the 3′-end of a Qprobe. When a Qprobe has hybridized with theinternal standard gene, a G does not present in a position complementaryto the fluorescence-labeled 5′-end of a Qprobe; as a result, it does notreceive any fluorescence-quenching effect. On the contrary, when aQprobe has hybridized with the target nucleic acid, it receives amarkedly fluorescence-quenching effect owing to the presence of a G. AQprobe can, therefore, specifically detect a target nucleic acid basedon a difference of fluorescence-quenching rates.

The change in fluorescence in FIG. 9B is contrary to that of FIG. A. Inother wards, a Qprobe receives a markedly fluorescence effect onhybridizing with an internal standard nucleic acid, which effect iscaused by the presence of a G of the internal standard nucleic acid in aposition corresponding to a C of the fluorescence-labeled 5′-end of theprobe; on hybridization with the target gene, a Qprobe is not found toreceive any markedly fluorescence-quenching effect because an A ispresent in a position of the target gene complementary to a C of thefluorescence-labeled 5′-end. A Qprobe can, therefore, specificallydetect an amplifying product from the internal standard gene.

Assaying Method 1-2 for Assaying Nucleic Acid (FIG. 2 Should beReferred)

It is a method for assaying a nucleic acid by determining a Tm value,which determination is conducted using a below model of a novel mixtureselected from the above-described mixtures comprising an internalstandard nucleic acid and a target nucleic acid probe. FIG. 2illustrates specifically the method.

a) Novel Mixture

1) It comprises one or two or more pairs of one or two or more types ofthe below target nucleic acid probes and one or two or more types of thebelow internal standard nucleic acids.

The target nucleic acid probe: Any target nucleic acid probe asdescribed above can be used, insofar as it is caused a fluorescentcharacter to change on the hybridization with the internal standardnucleic acid. In particular, it preferably has a portion (including aportion of one base) not complementary to the internal standard nucleicacid in one or two or more portions of the probe other than afluorescence-labeled portion.

The internal standard nucleic acid: in particular, it is a preferableone having a portion (including a portion of one base) not complementaryto the target nucleic acid in one or two or more portions of theinternal standard nucleic acid. Further, it is a preferable one having abase-sequence structure (including a structure of one base) such that aregion other than a region corresponding to a fluorescence-labeledportion of the target nucleic acid probe is different in one or two ormore portion in a region of the internal standard nucleic acidcorresponding to the hybridization region of the target nucleic acidprobe with the target nucleic acid.

In the present invention, the following instances are preferable.

1) A fluorescent dye-labeled portion of the target nucleic acid is aportion including a C or a G, or these bases (within a portion of fromthe 1st base to the 3rd base, the labeled is counted as the 1st base).

2) The target nucleic acid probe is a Q probe.

3) The labeled portion of the above 1) is the 3′-end or 5′-end.

b) The Method for Assaying a Nucleic Acid

1) This method is an illustrative method applicable when it is a methodfor assaying a target nucleic acid by determining a Tm value using theabove novel mixture. The method as described in the below 2) ispreferable.

2) The method is a method comprising adding one or plural target nucleicacids into the above-described novel mixture; conducting ahybridization; measuring a fluorescent intensity of the hybridizingsolution with increasing temperature; and determining the target nucleicacids by the below procedures.

Procedures:

(1) drawing a curve dependent to changed fluorescent intensity measured;

(2) differentiating the resulting curve;

(3) integrating the resulting peak(s) and determining the area(s) of thepeak(s);

(4) calculating a ratio(s) of the resulting peak area(s) of the internalstandard nucleic acid and the resulting peak area(s) of the targetnucleic acid;

(5) multiplying the concentration of the internal standard nucleic acidby said ratio.

Further, the present invention relates to kits comprising theabove-described nucleic acid probe and internal standard nucleic acid.

These are hereinafter described with reference of FIG. 2.

On dissociation of the hybridizing complex of a Qprobe and a targetnucleic acid, an interaction between a fluorescent dye and guanine iscancelled; the probe becomes to again emit fluorescence. A dissociationcurve can be rapidly and easily determined by monitoring fluorescence incontinuation with changing temperature; a dissociation peak can beobtained by differentiating the resulted dissociating curve. A peak arearatio of the dissociation peaks varies depending upon a constituentratio of genes (a existing ratio of target nucleic acids). This methodis related to a method for determining a gene-constituent ratio based onthe dissociation peaks.

This method comprises a processing step adding an internal standardnucleic acid; the usable internal standard nucleic acid is an internalstandard nucleic acid having mutation incorporated into the regionhybridizing with a QProbe. When there is no mismatching pair between aQProbe and the target nucleic acid, thereby, no mismatching pair becomesto be present between a QProbe and the internal standard nucleic acid.In this instance, the dissociation between the internal standard nucleicacid and a QProbe occurs in lower temperature than that between thetarget gene and a QProbe. When a target gene and an internal standardgene are present together, therefore, the dissociation curve becomessuch a curve that two curves have been fused. If a difference between aTm value of a hybridizing complex formed with an internal standard geneand a QProbe and that with an target nucleic acid and a QProbe isenough, the dissociation curve can be completely separated into two(FIG. 2 should be referred). A height ratio of the peaks is highlyco-relative to a gene-constituent ratio; a gene-constituent ratio amonggenes in a system can be determined based on a height ratio of peaksobtained from a dissociation curve. A target gene is, thereby,quantitatively assayed.

In this method, it can be mentioned, as the requirement meeting apreferable internal standard nucleic acid, that dissociation peaks in atarget gene and dissociation peaks in an internal standard nucleic acidare separated so that a gene ratio among genes in a system aredetermined. Therefore, this method is an illustrative method applicablewhen an internal standard gene meets the above requirement.

This method, like the method for assaying a gene using thebelow-described two QProbes (a target nucleic acid probe and an internalstandard nucleic acid probe), a method for assaying quantitatively atarget gene by determining a gene constituent ratio. Thereby, even whenthe concentration of a probe is lower than a total sum concentration ofa target gene and an internal standard gene, it is possible toquantitatively determine correctly a concentration of the target geneinsofar as the concentration of an internal standard gene is suitablefor determining a gene constituent ratio. On account of this, thismethod has following features: 1) no requirement for a processing stepfor diluting a target gene; 2) no requirement for changing aconcentration of a probe; it is possible to solve conventional problems.

Assaying Method 2

It is a method using the above-described doubly-labeled nucleic acidprobe; it will be illustrated with reference of FIGS. 3, 11, 12, 13, 14and 15.

Problems for a novel method for assaying a gene by using an internalstandard nucleic acid as described above.

i) Only two fluorescent dyes are known as a fluorescent dye such that itis different in fluorescent characteristics such asfluorescence-quenching effect by interaction with guanine, an exitingwavelength, and a fluorescence-emitting wavelength. It is impossible todetect at the same time two or more genes exiting in a homogenoussolution system. Because of this deficiency, if two or more genes areasked for detecting, there is no means except for increased number ofsamples; as a result, a method for assaying a gene using QProbe has beenexpensive.

ii) Two probes for a homogenous solution are used for a method forassaying a gene using two QProbes, which method is the above-describednovel method for assaying a gene. Each probe causes afluorescence-quenching effect on hybridization with any one of thetarget gene and the internal standard gene. In addition, a QP probeusing in this method can hybridize with a target gene and an internalstandard gene without differentiating both genes, on other wards, halfportions of the probe hybridizing with a target gene or an internalstandard gene causes no fluorescence-quenching effect. As a result, theresulting fluorescence-quenching rate is not great as an expected rate;in particular when a target gene is little in the amount, there areproblems such that analyzing errors become greater.

The foresaid problems can be solved by the above described methodaccording to the present invention. That is, the problems have beenexpected to be capable of being solved by each of the both ends of aQProbe being labeled with one sort of fluorescent dye being differentfrom the other dyes (see FIG. 3). By using one sort of doubly-labeledprobe (called a “Switching probe”) obtainable by doubly-labeling bothends of a QProbe with two sorts of dye different from each other, whenthe probe hybridizes with any of a target gene and an internal standardgene, a fluorescence-quenching effect is caused, namely, any of thehybridized probes causes a fluorescence-quenching effect. Owing to thisfluorescence-quenching effect, a fluorescence-quenching rate istheoretically enhanced two times or more as compared with that using twosorts of QProbes; as a result, a detecting sensitivity is possible toimprove two times or more (see FIGS. 3, 11, and 13).

This method is specifically not limited insofar as it is a method usinga novel mixture comprising the above described doubly-labeled nucleicacid probe and internal standard nucleic acid. Further, it can be amethod using the doubly-labeled nucleic acid probe and internal standardnucleic acid described above.

It is, however, preferable to use a novel mixture comprising the belowdoubly-labeled nucleic acid probe and internal standard nucleic acid, orto use the below doubly-labeled nucleic acid probe and internal standardnucleic acid.

The doubly-labeled nucleic acid probe: Any doubly-labeled nucleic acidprobe as described above is usable without any specified limitation. Ifchanges in optical characters of two sorts of fluorescent dyes labeledto the doubly-labeled nucleic acid probe fall within, as one example,the following instances, the probe is preferable.

1) It is a doubly-labeled nucleic acid probe such that, uponhybridization of the above nucleic acid probe with a target nucleicacid, the fluorescence of anyone of two labeled dyes is quenched, andthe other emits fluorescence; and in this instance, upon hybridizationwith an internal standard nucleic acid, the fluorescence-quenched dyeemits fluorescence and the fluorescence of the fluorescence-emitted dyeis quenched. The right drawing of FIG. 3 and FIG. 13 should be referredto. The base sequence of a probe corresponding to this instance can haveoptional bases as bases of both of the dye-labeled portions, but onother hand, the base sequence of a target nucleic acid is designed suchthat a portion of the target nucleic acid corresponding to a portionlabeled with any one of both dyes used in the probe includes a G, andthat corresponding to a portion labeled with the other dye does notinclude a G, but includes only an A and/or a T. The base sequence of theinternal standard nucleic acid is designed such that it is contrary tothat of the target nucleic acid.

2) It is a doubly-labeled nucleic acid probe such that, uponhybridization of the above nucleic acid probe with a target nucleicacid, the fluorescence of both of labeled dyes is quenched, or the bothemit fluorescence; and in this instance, upon hybridization with aninternal standard nucleic acid, the fluorescence of any one of both ofthe labeled dyes is quenched, and the other dye emits fluorescence; orin the instance contrary to the above, on hybridization with an internalstandard nucleic acid, the fluorescence of both of the labeled dyes isquenched, or the both emit fluorescence; and in this instance, uponhybridization with a target nucleic acid, the fluorescence of any one ofboth of the labeled dyes is quenched, and the other dye emitsfluorescence; See FIG. 13.

In the base sequence of the probe in an instance as above, as anillustrative base sequence, a base of a dye-labeled portion of the probecan be optional; a base sequence in a target nucleic acid side should bedesigned so that both regions of the target nucleic acid correspondingto both portions labeled with two dyes in the probe include a G, or doesnot a G. On other hand, a base sequence in the internal standard nucleicacid should be designed so that any one of both of the regions includesa G.

As an additional illustrative base sequence, a base of a dye-labeledportion of the probe can be optional; a base sequence in a targetnucleic acid side should be designed so that any one of both regions ofthe target nucleic acid corresponding to both portions labeled with twodyes in the probe include a G. On other hand, a base sequence in theinternal standard nucleic acid should be designed so that both of theregions include a G, or does not a G. See FIG. 13.

In the present invention, such a probe as described above is preferablyusable.

Internal standard nucleic acid: The above description of the abovedoubly-labeled nucleic acid should be referred to.

Incidentally, in this invention also, as a doubly-labeled nucleic acidis a Qprobe preferable. In addition, a characterizing form of the abovenucleic acid probe is preferably similar to that in the first inventionexcept the above-described characteristics.

The illustrative method for assaying a nucleic acid is described asfollows:

The following instance is a preferable method; this instance imposes nolimitation on the present invention.

It comprises: adding one or plural target nucleic acid into theabove-described mixture and conducting hybridizing reaction; measuring achange in an optical character from both dyes in the reaction system;calculating a ratio of the resulting measuring values, or a rate of anchanged optical character (one example: a fluorescence-quenching rate);and determining a concentration of the target nucleic acid. Anillustrative method will be described in examples 6 and 7.

Assaying Method 3

It is a method using a mixture obtained by adding further an internalstandard nucleic acid probe into the mixture as described in theforegoing (1). See FIG. 1.

Insofar as the method is a method using a novel mixture containing aninternal standard nucleic acid probe in addition to the ingredient inthe mixture as described above, any method can be applicable; thefollowing method is preferable.

The method is characterized, in that the method comprises: adding one orplural target nucleic acid into the above-described mixture andconducting hybridizing reaction; measuring a change in an opticalcharacter from each dye in the reaction system; calculating a ratio ofthe resulting measuring values, or a rate of changing in an opticalcharacter (a decreasing rate or a emitting rate); and determiningconcentrations of one or plural target nucleic acids.

The method for assaying a target nucleic acid according to the presentinvention is shown in FIG. 1, in which it uses a reaction solution priorto a reaction, including the simplest target nucleic acid probe,internal standard nucleic acid probe and internal standard nucleic acid.However, the present invention can not be limited by an illustrativemethod of FIG. 1.

In FIG. 1, a QProbe is used as a target nucleic acid probe or internalstandard nucleic acid probe for simplifying.

The method will be described hereinafter with reference to FIG. 1 byusing the simplest target nucleic acid probe, internal standard nucleicacid probe and internal standard nucleic acid. In a method shown in FIG.1, QProbe A is used as a target nucleic acid probe and QProbe B as aninternal standard nucleic acid probe.

Target Nucleic Acid Probe

It is designed such that it hybridizes indiscriminately a target gene(hereinafter, called a “target nucleic acid”) and an internal standardgene (hereinafter, called an “internal standard nucleic acid”); and onlyon hybridization with a target nucleic acid, a change (for example, thefigure shows decreasing (quenching)) in an optical character(fluorescence-emission) of dyes used for labeling the probe can occur.The probe is, in this instance, preferably designed such that the endregion opposite a labeled portion of the probe is not complementary to atarget nucleic acid. That the number of non-complementary bases of thetarget nucleic acid probe is caused to be equal to that of bases of theinternal standard nucleic acid probe, which bases are complementary tothe target nucleic acid in a fluorescence-labeled portion of theinternal standard nucleic acid is preferable because thethermo-stability of the hybridizing complex between a target nucleicacid probe and a target nucleic acid comes to be identical to thatbetween an internal standard nucleic acid probe and an internal standardnucleic acid.

That is, an fluorescent dye-labeled end portion of the probe is causedto be complementary to a region of the target nucleic acid correspondingto the fluorescent dye-labeled end portion of the probe and is caused tobe not complementary to the region of the internal standard nucleic acidcorresponding to that of the probe. On other hand, the end portionopposite the fluorescence-labeled portion is caused to be notcomplementary in the target nucleic acid and to be complementary in theinternal standard nucleic acid.

Specifically, a fluorescence-labeled portion of the probe is caused tobe a portion containing a G or a C, or to be a G or C self. A basesequence of the region of the target nucleic acid corresponding to thelabeled portion of the probe is designed such that the correspondingregion is caused to contain a C or a G, to be a C or G self. Thisdesigning means that the base of the target nucleic acid correspondingto the labeled base of the probe is not necessarily a C or a G; and atleast one base of a C or a G is included within a region (in a range ofone base to three bases in a direction of the 5′- or 3′-end apart fromthe base corresponding to a labeled base of the probe, wherein thecorresponding base is counted as the 1^(st) base) of the target nucleicacid containing the base corresponding to a labeled base of the probe.

On the other hand, a base sequence of the other end region of the probeshould be designed such that the base sequence is caused to be notcomplementary to a target nucleic acid, but to be complementary to aninternal standard nucleic acid. Since this region of the internalstandard nucleic acid corresponding to the other end region of the probehas been designed so as to be complementary to a fluorescence-labeledportion of an internal standard nucleic acid probe, the region includesat least one base of a C or a G. Accordingly, the other end region ofthe target nucleic acid probe includes at least one base of a C or a G,or is a portion of self thereof. The correspondence should be that theregion of the internal standard nucleic acid corresponding to the otherend region of the probe includes a G or a C, or is a portion of selfthereof; that of a target nucleic acid includes a C, or is a portion ofC self. On the contrary, when a corresponding region of an internalstandard nucleic acid includes a C, or is a portion of self thereof,that of a target nucleic acid should include a G, or should be a portionof G self. These correspondences are shown in FIG. 1.

Internal Standard Nucleic Acid Probe:

Its base sequence should be the same as that of a target nucleic aidprobe, wherein a fluorescence-labeled portion should be contrary to thatof the target nucleic acid probe as shown in FIG. 1. Further, afluorescent dye to be labeled should be different from that of thetarget nucleic acid. It is designed such that it hybridizesindiscriminately a target gene and an internal standard gene; and onlyon hybridization with a target nucleic acid, changes (for example, thefigure shows decreasing (quenching)) in optical characters(fluorescence-emission) of dyes used for labeling the probe can occur.The probe is, in this instance, preferably designed such that the endregion opposite a labeled portion of the probe is not complementary atarget nucleic acid. That the number of non-complementary bases of thetarget nucleic acid probe is caused to be equal to that of bases of theinternal standard nucleic acid.

Internal Standard Nucleic Acid

It is designed such that it is caused to hybridize indiscriminately atarget nucleic acid probe and an internal standard nucleic acid probe;and only on hybridization with an internal standard nucleic acid probe,changes in optical characters of dyes used for labeling the probe canoccur. Insofar as an internal standard nucleic acid meets thisrequirement, any internal standard nucleic acid with any structure ispreferably applicable. For example, the base sequence of the internalstandard nucleic acid is preferable to make identical to that of thetarget nucleic acid except both end bases.

It should be designed such that the base of the internal standardnucleic acid corresponding to the base of a fluorescent dye-labeledportion of an internal standard nucleic acid probe is complementary tothe base of the internal standard nucleic acid probe. On the other ward,when the labeled region of the internal standard nucleic acid probecontains a C, or is a portion of C self, the corresponding region of theinternal standard nucleic acid contains a or is a portion of G self; andwhen the labeled region of the internal standard nucleic acid probeincludes a G, or is a portion of G self, the corresponding region of theinternal standard nucleic acid contains a C, or is a portion of C self.

It should be designed such that the region of the internal standardnucleic acid corresponding to the base of a fluorescent dye-unlabeledportion of an internal standard nucleic acid probe is not complementaryto the base of the internal standard nucleic acid probe. That is, whenthe unlabeled region of the internal standard nucleic acid probeincludes a G, or is a portion of G self, the corresponding region of theinternal standard nucleic acid is caused not to contain a C, and iscaused to be a portion containing only a G, an A or a T, or to be aportion of each self thereof, preferably being caused to be a portionincluding only an adenine (a A) and/or a thymine (a T), or to be aportion of each self thereof. On the contrary, when a correspondingregion of an internal standard nucleic acid probe contains a C, or is aportion of C self, a corresponding region of an internal standardnucleic acid is caused not to contain a G, and is caused to be a portioncontaining only a C, an A, a T, or a portion of each self thereof.

By the above designing, the thermo-stability of the hybridizing complexbetween a target nucleic acid probe and a target nucleic acid comes tobe identical to that between an internal standard nucleic acid probe andan internal standard nucleic acid; both probes are expected to becapable of hybridizing with a target nucleic acid and an internalstandard nucleic acid indiscriminately.

In the following description, this assaying method will be illustratedwith reference to FIG. 1. On hybridization of QProbe A with a targetnucleic acid, a G of the target nucleic acid comes at a positioncomplementary to a fluorescence-labeled C (labeled with a fluorescentdye); the fluorescence emission of QProbe A is markedly quenched (afluorescent intensity is reduced) (hereinafter, meaning the same). Abase sequence of an internal standard nucleic acid is arranged such thatan adenine (a A) of the internal standard nucleic acid comes at aposition complementary to the 5′-end base C of QProbe A; as a result, onhybridization of QProbe A with the internal standard nucleic acid, thefluorescence-emission of Qprobe A is not quenched because G is notpresent in a position complementary to the fluorescence-labeled 5′-end.On hybridization with the target nucleic acid, the fluorescence-emissionof QProbe A is markedly quenched, because G comes at the complementaryposition: In this way, a QProbe can detect specifically a target nucleicacid based on the difference of quenching rates.

Further, on hybridization of QProbe B with the internal standard, thefluorescence-emission of QProbe B is markedly quenched, because G of thetarget nucleic acid comes at a position complementary to afluorescence-labeled 3′-end C; on hybridization of QProbe B with thetarget nucleic acid, the fluorescence-emission of Qprobe B is not foundto be quenched because an A comes at a position complementary to thefluorescence-labeled 3′-end C of the QProbe B. In this way, QProbe B candetect, on the contrary to QProbe A, specifically a PCR-amplifiedproduct from the internal standard nucleic acid.

Method for Assaying Target Nucleic Acid

Since the novel mixture according to the present invention comprises theabove-described constituents, the assaying method comprises: adding oneor plural target nucleic acid into the mixture and conductinghybridizing reaction; measuring a change in an optical character fromthe target nucleic acid probe and a change in an optical character fromthe internal standard nucleic acid probe; calculating a ratio of theresulting measuring values, and determining a concentration of thetarget nucleic acid by multiplying a used concentration of the internalstandard by an obtained calculating value.

That is, each of the above-described QProbe A and QProbe B can hybridizewith the target gene and the internal standard gene indiscriminately.Thereby, the hybridization of any of the probes with any of genes reliescompletely on an existing ratio of genes. On this reason, a ratiobetween measuring values obtained by measuring changes in opticalcharacters from QProbe A and that from QProbe B becomes to be aconstituent ratio between the target nucleic acid and the internalstandard nucleic acid.

The novel method for assaying a target nucleic acid is, as describedabove, a method for determining a gene constituent ratio. Thereby, evenwhen the total sum concentration of a target nucleic acid and aninternal standard nucleic acid is higher than a total sum concentrationof a target nucleic acid probe and an internal standard nucleic acidprobe (such case as that a nucleic acid concentration is higher than aprobe concentration), it is possible to determine a constituent ratio ofa target nucleic acid and an internal standard nucleic acid insofar asthe concentration of an internal standard nucleic acid is suitable fordetermining a nucleic acid constituent ratio (insofar as a concentrationof an internal standard nucleic acid is enough close to a concentrationof a target nucleic acid). As a result, this method can accuratelydetermine quantitatively a concentration of a target nucleic acid. Onaccount of this, in this method, 1) it is not necessary to dilute asolution containing a target gene, and 2) it is not required to changean additive concentration of a probe, because the additive concentrationof a probe can be adjusted at the lowest concentration fallen in aconcentration range detectable by an instrument used forfluorescence-detecting. It is concluded that this method has followingfeatures: 1) no requirement for a processing step for diluting a targetgene; and 2) no requirement for changing a concentration of a probe; itis possible to solve conventional problems.

In an additional aspect of the present invention, there is provided thefollowing probes and kits.

1) One or plural pairs of nucleic acids comprising the above-describedtarget nucleic acid probes and internal standard nucleic acid probes.

2) Kits for assaying a target nucleic acid, comprising one or pluralpairs of nucleic acids comprising the above-described target nucleicacid probes and internal standard nucleic acid probes; and one or pluralinternal standard nucleic acids corresponding to pairs of the nucleicacid probe as described in above 1).

Assaying Method B:

The method has the following two ways.

The method is based on hybridization without discriminating a targetgene and an internal standard gene; that any of the probes hybridizeswith any of genes relies completely on an existing ratio of genes. Owingto this, for example, it is expected that a constituent ratio of genescan be determined, based on hybridizing ratio between the hybridizationof QProbe A with a nucleic acid and the hybridization of QProbe B with anucleic acid. Fluorescence-quenching is excitingly caused onhybridization of QProbe A with a nucleic acid or on hybridization ofQProbe B with an internal standard nucleic acid. On this reason, a ratiobetween a fluorescence-quenching rate from QProbe A and that from QProbeB is positively proportional to a constituent ratio between the targetnucleic acid and the internal standard nucleic acid; as a result, aconstituent ratio of genes can be determined based on the ratio offluorescence-quenching rates.

The method will be explained below.

Assaying Method 1

It is a novel method for assaying a nucleic acid, wherein the novelmixture according to the present invention comprises any one of thefollowing target nucleic acid probe, doubly-labeled nucleic acid probe,and multiply-labeled nucleic acid probe and the predetermined amount ofan internal standard nucleic acid. Hereinafter, these will be called“recognizable nucleic acid probe”. The novel mixture in this methodcomprises one or two or more nucleic acids as described below. In thenovel mixture comprising two or more nucleic acids, sorts of dyes, withwhich each probe is labeled, are different from each others; and thedyes have different optical characters from each others, as describedabove.

Novel Mixture

It comprised one or two or more nucleic acids and internal standardnucleic acids as described below.

Target Nucleic Acid Probe

The target nucleic acid probe of the present invention is notcomplementary to a target nucleic acid and an internal standard nucleicacid at one or two or more portions of a fluorescent dye-labeled region.

Doubly-Labeled Nucleic Acid Probe

The doubly-labeled nucleic acid probe according to the present inventionis not complementary to any one of a target nucleic acid and an internalstandard nucleic acid at a labeled portion with any of two dyes used forlabeling the doubly-nucleic acid.

The method according to the present invention can, subsequent todetermination of an existing ratio of a target nucleic acid and aninternal standard nucleic acid, determine correctly a concentration oramount of the target nucleic acid based on the obtained existing ratio.

Multiply-Labeled Nucleic Acid Probe

It is a probe to which the described doubly-labeled probe is applied. AnA, a C or a G containing portion (preferably C) of region one (1) toregion n of a single stranded oligonucleotide (including an end portion,a base moiety, a sugar moiety, or a phosphate moiety of a portion in itschain; n stands 10, preferably 5, more preferably 3.) is labeled witheach of dye 1 to dye n, respectively, which each dye can cause a changein an optical character on its hybridization with a target nucleic acid.Sorts of dye 1 to dye n each independently are different fluorescentdyes. In addition, region 1 to region n independently are differentregions. The structure of a labeled portion in the probe and thestructure of a target nucleic acid corresponding to the probe are likethose as described above.

Incidentally, a preferable example of the multiply-labeled nucleic acidprobe is a QProbe; further, its preferable labeled portion is a portionof C self or a region containing the portion.

Internal Standard Nucleic Acid

It is a nucleic acid like the internal standard nucleic acid used in theassaying method using a doubly-labeled nucleic acid probe. Further, asdescribed in the above, the structure of its portion corresponding toeach of the fluorescent dye-labeled portions of the multiply-labelednucleic acid probe should be different from that of a nucleic acid. The“different structure” herein means whether or not a structure iscomplementary to a fluorescent dye-labeled portion of the probe. Thesestructures each have a structure applied correspondingly to the aboveboth structures. In the case of two or more target nucleic acids, two ormore internal standard nucleic acids become to be neededcorrespondingly; the above-described novel mixture of the presentinvention should comprise two or more internal standard nucleic acids.

Assaying Method

Its method comprises: adding one or two or more target nucleic acidsinto the novel mixture in this method; conducting hybridization;measuring a change or changing rate (for example, a quenching rate) inan optical character of each fluorescent dye labeled to each probe inthe hybridizing system using one or two or more measuring wave length,in that the change is caused by the hybridization of a target nucleicacid or internal standard nucleic acid and a corresponding probe;calculating a ratio between a measuring value in regard to the targetnucleic acid and that in regard to the internal standard nucleic acid;and determining one or two or more target nucleic acids on the basis ofthe obtained ratio because the concentrations of the internal standardnucleic acids have been predetermined.

Assaying Method 2

It is a method based on the determination of a Tm value.

A usable novel mixture may be an one like the mixture as describedabove.

Assaying Method:

The method is a method comprising adding one or plural target nucleicacids into the above-described novel mixture; conducting ahybridization; measuring a fluorescent intensity of the hybridizingsolution with increasing temperature; and determining a existing ratioof the target nucleic acids by the below procedures.

Procedures: (Conducting this Operation in Every Probes)

(1) drawing a curve dependent to a change in an optical charactermeasured;

(2) differentiating the resulting curve;

(3) measuring the height value(s) of a resulting peak(s), or integratingthe resulting peak(s) and determining the area value(s) of the peak(s);

(4) subsequent to election of a peak (a referential peak of a targetnucleic acid), calculating a ratio(s) of the resulting peak heightvalue(s) or area value(s) of the internal standard nucleic acid to theresulting peak height(s) or area(s) of the target nucleic acid (the peakheight value of an other target nucleic acid/the referential peak heightvalue, or the peak area value of another target nucleic acid/thereferential peak area value);

(5) calculating a existing ratio of the referential nucleic acid to thecomparative nucleic acid;

(6) determining a concentration or amount of the target nucleic acid onthe basis of the obtained existing ratio, because the concentration ofthe internal standard nucleic acid has been predetermined.

The above method will be illustrated using a QProbe.

On dissociation of the hybridizing complex of a Qprobe and a targetnucleic acid, an interaction between a fluorescent dye and guanine iscancelled; the probe becomes to again emit fluorescence. Thereby, adissociation curve can be rapidly and easily determined by monitoringfluorescence in continuation with changing temperature; a dissociationpeak can be obtained by differentiating the resulted dissociating curve.A peak area ratio of the dissociation peaks varies depending upon aconstituent ratio of genes (a presence ratio of target nucleic acids).This method is related to a method for determining a gene-constituentratio based on the dissociation peaks.

This method is a method for determining a existing ratio of genes.Thereby, even when the total sum concentration of a target gene and aninternal standard gene is higher than a total sum concentration ofQProbe A and QProbe B (such case as that a gene concentration is higherthan a probe concentration), it is possible to determine a constituentratio of a target gene and an internal standard gene insofar as theconcentration of an internal standard gene is suitable for determining agene constituent ratio (insofar as a concentration of an internalstandard gene is enough close to a concentration of a target gene). As aresult, this method can determine accurately and quantitatively aconcentration of a target gene.

On account of this, in this method, 1) it is not necessary to dilute asolution containing a target gene, and 2) it is not required to changean additive concentration of a probe, because the additive concentrationof a probe can be adjusted at the lowest concentration fallen in aconcentration range detectable by an instrument used forfluorescence-detecting. It is concluded that this method has followingfeatures: 1) no requirement for a processing step for diluting a targetgene; and 2) no requirement for changing a concentration of a probe; itis possible to solve conventional problems.

Assaying Method C:

In the above method, this method comprises assaying a target nucleicacid by quenching the fluorescence from a target nucleic acid probe orinternal standard nucleic acid probe not hybridizing with either aninternal standard nucleic acid or a target nucleic acid. An object ofthe present invention is capable of being achieved by using illustrativequenching substances or quenching substance-labeled probes as describedbelow. These are mere examples; these cannot impose any limitation onthe present invention. See Example 9.

Quenching substance: Illustrative are the above quenchers. Any one ofthe illustrative substances can be preferably used. Preferablyillustrative are Dabcyl, BH, Eclipse, Elle Quencher.

Quenching substance-labeled probe: Preferably usable is a probe labeledwith any one of the above quenchers at a portion of an oligonucleotide(any of a deoxynucleotide body and ribonucleotide body can be usable).Specifically see Example 9. As a labeling portion or position can bepreferably usable any portion or position insofar as those can cause achange in an optical character (for example, a fluorescence emission orquenching) of a target nucleic acid probe and/or internal standardnucleic acid probe not hybridizing with a target nucleic acid and/or aninternal standard nucleic acid. In particular, there is no limitation inthis instance.

Assaying Method D:

It comprises, subsequent to adding an exonuclease into an assayingsystem of the present invention specifically upon hybridization reachingto equilibrium, measuring a change in an optical character of theassaying system before and after the addition. See Example 10. Thehybridization with equilibrium induces a change (for example,fluorescence emission or quenching) in the optical character(s) of atarget nucleic acid probe and/or an internal standard nucleic acid. Theaction of the exonulease to the probe (s) under such conditions can makea mononucleotide labeled with a fluorescent dye free from the nucleicacid probe(s). The free mononucleotide labeled with a fluorescent dyedoes not any longer express any specific optical characteristicsmanifested on hybridization of a nucleic acid probe with a nucleic acid.This method measures that change. The specific method for assaying anucleic acid will be described in Examples.

Incidentally, preferable conditions for assaying are described in thefollowing.

Conditions: The labeled target nucleic acid probe and/or internalstandard nucleic acid probe are (is) not complementary to at least oneof the nucleic acid and internal standard nucleic acid in a regioncontaining a fluorescence-labeled portion (the region: a sequence of onebase to three bases, preferably of one base).

A novel mixture according to the present invention contains an enzyme;the mixture containing the enzyme is not in a form such that the enzymeis mixed with a mixture comprising a target nucleic acid probe and/or aninternal standard nucleic acid probe and an internal standard nucleicacid, but the enzyme is preferably in a form attached at a set to thenovel mixture.

Examples of usable exonucleases: (however, the examples impose nolimitation on the present invention.)

1) 3′→5′Exonuclease

Exonuclease I (Armersham Biosciene Corp.), Vent DNA polymerase (NewEngland Biolabs), T7 DNA polymerase (New England Biolabs), KlenowFragment DNA polymerase (New England Biolabs), Phi29 DNA polymerase (NewEngland Biolabs), Exonuclease III (Fermendas).

2) 5′→3′ Exonuclease

Taq DNA polymerase, Exonuclease VII (Armersham Biosciene Corp.)

3) Other Usable Enzyme

S1 Nuclease (Armersham Biosciene Corp.), Mung Bean Nuclease (ArmershamBiosciene Corp.).

Third Invention

It is an invention wherein the above-described inventions are combinedwith a nucleic acid-amplifying method. In this invention, a productamplified by the nucleic acid-amplifying method is assayed by using theabove method for assaying for assaying a nucleic acid. This method isapplicable to any product amplified by a gene-amplifying method. Thismethod comprises, in particular, amplifying one or plural target nucleicacids in a reaction system of the above invention containing one orplural internal standard nucleic acids by a gene-amplifying method;assaying a nucleic acid in the obtained reaction solution or amplifiedproduct as a sample by using the above-described nucleic acid-assayingmethod.

The invention comprises inventions A, B, C and D.

1. Invention A

1) This method comprises, in particular, amplifying one or plural targetnucleic acids in a reaction system comprising one or plural internalstandard nucleic acids until a stationary phase (including an initialphase, middle phase and stationary phase) by a gene-amplifying method;and assaying a nucleic acid in the obtained reaction solution oramplified product as a sample by a conventionally-known nucleicacid-assaying method.

2) The method comprises; assaying a nucleic acid in the amplifiedproduct as described in the above 1) as a sample by a novel nucleicacid-assaying method of the present invention.

3) This method comprises amplifying one or two or more nucleic acids ina reaction system comprising an internal standard nucleic acid of thepresent invention by a gene-amplifying method; and assaying one or twoor more nucleic acids prior to the amplification.

4) In the method for assaying a nucleic acid according to any one of theabove 1) to 3), the gene-amplifying method is a nucleic acid-amplifyingmethod using the same primer as a primer of a target nucleic acid and aprimer of an internal standard nucleic acid; and the method for assayinga nucleic acid is a method for assaying one or two more nucleic acidsprior to an amplification.

5) In the method for assaying a nucleic acid according to the above 3)or 4), the gene-amplifying method is a method using a Qprobe as aprimer; and the method for assaying a nucleic acid is a method forassaying one or two more nucleic acids prior to an amplification.

6) In the method for assaying a nucleic acid according to any one of theabove 3) to 5), the gene-amplifying method is a method using a primerwhich is caused an optical character of the primer to increase uponhybridization of the primer with a target nucleic acid and/or internalstandard nucleic acid; and the method for assaying a nucleic acid is amethod for assaying one or two more nucleic acids prior to anamplification.

This invention is applicable to any product obtained by agene-amplifying method. In particular, it is preferably applicable toamplified product of a gene, which makes it impossible to assay, thatis, the amplified product of a gene amplified until a stationary phase(including a stationary phase).

Invention B

1) It is related to a method for assaying a target nucleic acid, whereinsaid method comprises: amplifying one or two or more target nucleicacids by a gene-amplifying method in a reaction system comprising one ortwo or more internal standard nucleic acid, which is used in a methodfor assaying one or two or more target nucleic acids by a method fordetermining a Tm value as described in the above; and assaying one ortwo or more target nucleic acids using the obtained amplified product asa sample by a conventional method.

2) It is a method for assaying one or two or more target nucleic acidsprior to the amplification of the target nucleic acids using the abovesample by a method for determining a Tm value according to the presentinvention.

3) It is a method for assaying a target nucleic acid according to theabove 2), wherein said method is a method for assaying one or two ormore target nucleic acids prior to the amplification of the targetnucleic acids.

4) It is a method for assaying a target nucleic acid according to theabove 3), wherein said method comprises amplifying a target nucleic acidby using the same primer as a primer of the target nucleic acid and as aprimer of an internal standard nucleic acid.

5) It is a method for assaying a target nucleic acid according to theabove 4), wherein the using primer is Qprobe.

6) It is a method for assaying a target nucleic acid according to theabove 4), wherein the change in an optical character is an increase.

This invention is applicable to any product obtained by agene-amplifying method. In particular, it is preferably applicable toamplified product of a gene, which makes it impossible to assay, thatis, the amplified product of a gene amplified until a stationary phase(including a stationary phase).

Invention C

1) It is a novel method for assaying a target nucleic acid, wherein saidmethod comprise amplifying one or plural target nucleic acids in areaction system comprising one or plural internal standard nucleic acidsas described in invention 2 of the 2^(nd) invention; assaying one or twoor more target nucleic acids prior to the amplification of the nucleicacids using the obtained reaction solution or amplified product as asample, or

2) It is a novel method for assaying a target nucleic acid, wherein saidmethod comprise amplifying one or plural target nucleic acids in areaction system comprising one or plural internal standard nucleic acidsas described in invention 2 of the 2^(nd) invention; assaying using anovel mixture according to any one of the above 1) to 3) of invention 2of the 2^(nd) invention one or two or more target nucleic acids prior tothe amplification of the nucleic acids using the obtained reactionsolution or amplified product as a sample, or,

3) It is a novel method for assaying a target nucleic acid, wherein saidmethod comprise amplifying one or plural target nucleic acids in areaction system comprising the internal standard nucleic acids asdescribed in invention 2 of the 2^(nd) invention until a stationaryphase (including a stationary phase); assaying using the novel mixtureaccording to any one of the above 1) to 3) of invention 2 of the 2^(nd)invention one or two or more target nucleic acids prior to theamplification of the nucleic acids using the obtained reaction solutionor amplified product as a sample, or

4) It is a method for assaying one or plural target nucleic acids priorto the amplification of the target nucleic acids according to any one ofthe above 1) to 3), wherein said method comprises using the same primeras a primer of the target nucleic acid and as a primer of the internalnucleic acid, or,

5) It is a method for assaying one or plural target nucleic acids priorto the amplification of the target nucleic acids according to the above4), wherein said primer is a Qprobe, or,

6) It is a method for assaying one or plural target nucleic acids priorto the amplification of the target nucleic acids according to the above5), wherein the change in an optical character is an increase uponhybridization of the primer with a target nucleic acid and/or aninternal standard nucleic acid.

This invention is applicable to any product obtained by agene-amplifying method. In particular, it is preferably applicable toamplified product of a gene, which makes it impossible to assay.

Assaying Method D, END POINT-Assaying Method

Problems of a Gene-Assaying Method Through an Gene-Amplifying Method

The gene-assaying method through an gene-amplifying method is a methodcomprising amplifying a gene and determining the amplified gene; it ishighly sensitive and can assay even a mere-existing gene. Thereby, themethod is one of a gene-assaying methods used at present the mostpopularly. In the gene-assaying method through a gene-amplifying method,various methods are known; and those have problems such as describedsubsequently. These detailed contents will be mentioned below.

(1) Problems in Conventional Methods

i) Problem 1: A Real-Time Quantitative PCR Method

In a PCR method that is a gene-amplifying method used the most widely atpresent, the reaction is known to proceed in a exponential functionuntil the amplified product accumulates to a certain level. Withincreasing an amplified product, however, its amplification efficiencyis lowered and reaches to a fixed level (reaching to a plateau); thisindicates that the obtained amplified product is constant regardless theinitial amount of a gene on enough amplifying reaction. Thereby, theinitial amount of a gene is incapable of being assayed based on theamount of the amplified gene product after completed amplification (atan endpoint). This problem is common to a known gene-amplifying method,a PCR method being not restricted.

However, a gene-amplification efficiency of a common gene-amplifyingmethod is constant at an initial phase of the reaction regardless theinitial amount of a gene. Therefore, a cycle number of the reaction, inthat the amplified product reaches to a threshold, varies on initialamounts of a gene insofar as an amplified product is increased in anexponential function; for example, upon amplification of a predeterminedgene diluted in some diluting rates, the cycle number (a CT value) on anamplified product reaching to a threshold is in reverse correlation toan initial concentration of the gene.

This is, as described in the former, due to a fact that an amplificationefficiency of an initial phase is fixed regardless various amounts ofthe gene. A relative equation obtained in thus way is usable as acalculation curve for assaying the unknown amount of a target nucleicacid in an unknown sample. In a usual case, an amplification efficiencyof a target gene in an unknown sample is expected to be constant; theinitial concentration of the target gene can be determinedquantitatively from the above calculating curve, following thedetermination of a Ct value obtained in a similar manner as above.Accordingly, an initial concentration of a gene can be assayed insofaras an amplified product can be monitored in real time.

The gene-assaying method based on this principal is a real timequantitative PCR method. This method does not requires (1) post-PCRprocessing steps and (2) to open a reaction tube, so that this methodhas excellent features such that the assaying is simple and rapid; andthe content of the tube is not contaminated with a foreign matter. Onthe other hand, however, since an amplification process is necessary tomonitor in real time, this method embrassed problems such that (1) theapparatus is rendered larger and expensive; and (2) since the method isa method based on an assumption that the amplification efficiency of thegene used for preparing a calculation curve is the same as that of thegene in an unknown sample, thereby, if there is an inhibitor against agene amplification in an unknown sample, this assumption is capable notto be established, that is, it is difficult to obtain an appropriatevalue always in this method; (3) since the amplification process isnecessary to monitor in real time, one assaying instrument comes to beoccupied during assaying. Therefore, its sample-treating power isnaturally limited.

ii) Problem 2 in a Conventional Method: a Competitive PCR Method

As a method for assaying a target nucleic acid in high sensitivity, acompetitive PCR method is known commonly. This method comprises: addinga primer for amplifying a target gene and the target gene of thepredetermined amount (called an internal standard gene) for beingamplified by the same primer as in the target nucleic acid into areaction system; amplifying at the same time together the target geneand internal standard nucleic acid; subsequent to completedamplification, isolating and quantitatively determining the amplifiedproduct from the target gene and the amplified product from the internalstandard gene by using an electrophoresis and the like; calculating aratio of the amplified products (the amplified product from the internalstandard gene/that from the target gene); and determining aconcentration of the target gene by multiplying the amount of theinternal standard gene added at the start of amplification by theobtained ratio, because the amplification efficiency of the target geneand that of the internal gene is expected to be the same owing to theamplification using the same primer as a primer for the target gene andas that for the internal standard gene.

This method is difficult to be affected by an amplification inhibitorbecause a target gene and an internal standard gene are amplified in thecoexistence thereof; the reliance on the obtained determining value ishigh (its cause: for example, even if the inhibitor is present in aassaying system, an amplification efficiency is at all lowered likely inregard to a target gene and a internal standard gene; the existinginhibitor does not affect a ratio of amplified products; as a result, anaccurate determining value is obtainable in the competitive PCR.). Thecharacteristics can be mentioned such that the instrument for assayingis comparatively simple and non-expensive.

However, in this method, problems are pointed out such that (a)subsequent to completed gene-amplification, the amplified product isnecessary to assay, so that it results in troublesome and time-expensiveoperation; (b) on the determination of an amplified product, a reactiontube is necessary to be open, and owing to the opening, the reactionsolution has a possibility of contamination with a foreign amplifiedproduct; and (c) the operation of this method is troublesome and isdifficult to be automated, so that its sample-treating power is lower.

iii) Problem 3 in Conventional Method: an Real Time Competitive PCRMethod.

The real time competitive method is a method for assaying an initialconcentration of a target gene prior to the amplification of the targetgene, wherein it is an improved competitive PCR method and comprises:conducting an amplification of a target gene and an internal gene in thecoexistence thereof using a TagMan probe, a QProbe or the like;monitoring at the same time in real time an amplified product from thetarget gene and an amplified product from the internal standard gene;and determining an initial concentration of a target gene prior to theamplification of the target gene based on the fluorescent signal amountfrom each the amplified products from the target gene and internalstandard gene.

This method embraces excellent features such that it does not requires(1) post-PCR processing steps and (2) to open a reaction tube, and (3)further it is, however, difficult to be affected by an inhibitor becauseof a competitive method. There are problems like the real timequantitative PCR method in this method such that (i) the apparatususable for assaying is larger and expensive, and (ii) itssample-treating power is lower, because it is required in this method tomonitor at the same time in real time an amplified product from aninternal standard gene and that from a target gene. The problems asdescribed above are summarized in the following table. The conventionalgene-assaying method is recognized to have some problems. TABLE 1Problems in conventional gene - assaying methods Real time Real timeQuantitative Competitive Competitive Preferable Problems PCR method PCRmethod PCR method method Rapidity and ◯ X ◯ ◯ simplicity of operationPrevented ◯ X ◯ ◯ contamination with amplification product Affection byX ◯ ◯ ◯ inhibitor Expensiveness X ◯ X ◯ of apparatus Sample - X X X ◯treating power◯: Meeting for requirementX: Non - meeting for requirement

Incidentally, the above described gene-assaying methods are applicableto any gene-assaying methods other than a PCR method such as an NASBAmethod, an LAMP method, an RCA method, an ICAN method; the methods areat present applied to many gene-determining method through variousgene-amplifying method. These methods embraces problems at all common togene-amplifying methods; even in any gene-assaying method through anygene-amplifying method, the above-described problems are desired to besolved.

(2) Strategy for Solving these Problems

As described above, it is possible to assay an initial concentration ofa target gene prior to the amplification of the target gene based on aratio of an amplified product from a target gene and that from aninternal standard gene. If this ratio of amplified products are capableof being assayed in a rapid and simple way without opening a reactiontube after completed gene-amplification, (1) it does not require a PCRpost processing step and can simply, easily and rapidly assay a targetgene; (2) it is not needed to open a gene-amplifying reaction tube, sothat it does not have any risk of contamination with foreign amplifiedproducts; (3) the assay is difficult to be affected by inhibitorsbecause the method is a competitive method; (4) since a gene-amplifyingprocessing-step and a detecting processing step of a amplified productcan be completely divided, a large quantity-sample becomes to be capableof being treated, so that a sample-treating power can be convenientlyand non-expensively improved (for example, subsequent to thegene-amplification by using plural non-expensive PCR-apparatuses nothaving any fluorescence-measuring function, it comes to be possible totreat large quantitative-samples by analyzing obtained data in order,for example, even using only one fluorescence-measuring instrument); 5)it is possible to assay a target gene by using a markedly simple andnon-expensive measuring instrument because the amplifying processing ofa nucleic acid is not necessary to monitor in a real time way and themeasuring instrument is not requisite for having a thermally-cyclingfunction indispensable to a PCR, so that it is possible to assay a geneby a very concise and non-expensive instrument. This gene-assayingmethod is expected to be capable of becoming an excellent gene-assayingmethod with such characteristics as above.

A conventional competitive PCR method makes it possible to determine aratio of amplified products at an end point, but a reaction tube isnecessary to open; so the ratio is capable of being rapidly determined.A real time competitive PCR method, being conventional like the abovemethod and using a fluorescence-labeled probe, does not require to opena reaction tube and is capable of detecting rapidly an amplifiedproduct. A ratio of fluorescent signals obtained using a conventionalfluorescence-labeled probe in that method, however, does not alwaysreflect usually a quantitative ratio of amplified products; namely, aregional gene reflecting correctly it is limited. In order to determinea ratio of amplified products, each of amplified products requires to bedetected in a regional gene where a fluorescent signal ratio isquantitatively proportional to an amplified product ratio. Thereby, areal time competitive PCR method has to be always monitored. As statedabove, no method is present until now for determining an amplifiedproduct ratio at an endpoint and rapidly with closing a reaction tube.

In view of the above situation, the inventors have proceeded to examinethose problems to lead the discover of a novel method enabling it todetermine an amplified product ratio at an end point. This invention isrelated to a novel method for assaying a gene and a novel nucleic acidprobe usable therefore. In the following description, those details willbe described.

i) An End Point Assaying Method for a Gene Using a QProbe

Specific Detection of a Target and Internal Standard Nucleic Acid by aQProbe

It is an object of certain embodiments of the present invention toprovide with a method for detecting an amplified product using the abovedescribed doubly-labeled QProbe (a Switching probe) or two QProbes. Thisdescription is an embodiment of a determining method using a Switchingprobe. The Switching probe has features such that (1) the base of eachof both ends of the probe is a C, and (2) the both ends are labeled withdyes having colors different in fluorescent emission. The base sequenceof a target nucleic acid is designed such that a G is present at aposition complementary to a C of the 5′-end of the Switching probe, andan A is present at a position complementary to a C of the 3′-end; on theother hand, in the base sequence of an internal standard gene, the aboveG is replaced by an A, and the above A by G. Under such a situation, onhybridization of the Switching probe with an amplified product amplifiedfrom the internal standard gene, the absence of a G at a position of theamplified product corresponding to its 5′-end does not result in amarkedly fluorescence-quenching effect in the dye labeled at its 5′-end;the presence of G complementary to its 3′-end does result in.

On the other hand, on hybridization with an amplified product from thetarget gene, the presence of G at a position of the amplified productcorresponding to its 5′-end results in a markedly fluorescence-quenchingeffect in the dye labeled at its 5′-end; the absence of G complementaryto its 3′-end does not result in. In conclusion, each of the amplifiedproducts comes to be capable of specifically being detected by theobservation of fluorescence-quenching of any dye labeled at any the bothends.

Features of Internal Standard Gene

The base sequence of the internal standard gene has features such that(1) the internal standard gene is capable of be amplified by using aprimer common to that of a target gene, (2) a GC content of a targetgene is the same as that of the internal standard gene, (3) a length ofthe base sequence of a portion of the internal standard gene completelycomplementary to the target gene on hybridization with an amplificationproduct amplified from the target gene is the same as that of the targetgene complementary to the internal standard gene on hybridization withan amplification product amplified from the internal standard gene, and(4) a base sequence of the target nucleic acid is the same as that ofthe internal standard gene except the bases complementary to both endsof a QProbe.

Based on the above description, it is considered that there is nodifference between an affinity of the Switching probe to anamplification product from a target gene and that from an internalstandard gene. In conclusion, a Switching probe can hybridize at randomwith an amplification product from a target gene and that from aninternal standard gene without differentiating those; a ratio betweenSwitching probe binding to an amplification product from a target geneand Switching probe binding to that from an internal standard gene iscompletely identical to a ratio of an amplification product from thetarget gene and that from the internal standard gene. Thereby, Based ona ratio between fluorescence-quenching rates of the two dyes labeled toboth ends of the Switching probe, a ratio of amplification products canbe determined.

In addition, it is understood that a gene-amplification rate is the samein a target gene and internal standard gene because (1) a primer of atarget gene and internal standard gene is common, (2) a GC content of atarget gene is completely the same as that of the internal standardgene, and (3) the base sequences of a target gene and a internalstandard gene are the same except that two portions are different.Further, based on the above (2) and (3), no bias generated by re-bondingof amplification products could occur. Therefore, a constituent ratio ofgenes comes to be capable of keeping having a starting ratio. However,as requirements for an internal standard gene's presence in an assaysystem, the subsequent two matters will be described: (1) a startinggene ratio prior to gene-amplification is kept in any phase of theamplification (an amplification efficiency of an internal standard geneis the same as that of a target gene.), (2) a probe making use iscapable of hybridizing at random with an amplification product from atarget gene and that from an internal standard gene withoutdifferentiating those. Insofar as an internal standard gene meets theserequirement, an internal standard gene may or may not be required tohave the above features.

As described above, an amplification product can be quantitativelydetermined based on fluorescence-quenching rates of two dyes labeled tothe both ends of a Switching probe.

From the above description, a ratio of amplification products can bequantitatively determined by using an internal standard gene and aSwitching probe, wherein said determination can be rapidly and simplycarried out at an end point without opening a reaction tube; manysamples can be treated.

A method for assaying quantitatively a gene is a method comprisingamplifying a target gene; then determining a ratio of amplificationproducts from a target gene and an internal standard gene; and makingquantification of the target gene. Insofar as a method for amplifying agene is capable of amplifying together a target gene and internalstandard gene with keeping a starting gene-constituent ratio, the methodis applicable to any method for amplifying a gene regardless of itssort. Thereby, this method is applicable to an LAMP method, an RCAmethod, an ICAN method and the like, and further, a method foramplifying a gene other than a PCR method.

ii) End Point Gene-Determining Method by a dissociation curve analysismethod using a QProbe.

As mentioned above, a gene-constituent ratio can be determined byanalyzing a dissociation curve obtainable by a QProbe; this method isexpected to be applicable to a competitive gene-assaying method througha gene-amplifying method.

An internal standard gene usable in this method should be a probe suchthat a mutation is inserted into a portion of said probe capable ofhybridizing with a QProbe. Subsequent to completed amplification of agene, a dissociation curve is prepared of the obtained amplificationproducts using the QProbe, followed by preparation of dissociation peaksbased on the obtained dissociation curve. A gene-constituent ratio canbe determined based on a peak-height ratio of the dissociation peaksbecause the peak-height ratio is highly co-relative to agene-constituent ratio. It becomes possible to determine a concentrationof a target gene, subsequent to co-amplification of both of an internalstandard gene of a predetermined concentration—the gene-amplificationefficiency of the internal standard gene being the same as that of thetarget gene—and the target gene—using various gene-amplification method,by the determination of a ratio of an amplification product from atarget gene to an amplification product from the internal standard gene.

In this method, as requirements for an internal standard gene, tworequirements can be mentioned such that (1) a starting gene ratio priorto gene-amplification is kept in any phase of the amplification (anamplification efficiency of an internal standard gene is the same asthat of a target gene.), and (2) a dissociation peak derived from atarget gene are enough separated from a dissociation peak derived froman internal standard gene so that a gene-constituent ratio can bedetermined. Insofar as an internal standard gene meets theserequirement, an internal standard gene is not always required to havethe above features.

Insofar as a method for amplifying a gene is capable of amplifyingtogether a target gene and internal standard gene with keeping astarting gene-constituent ratio, the method is applicable to any methodfor amplifying a gene regardless of its sort. Thereby, this method isapplicable to an NASBA method, an LAMP method, an RCA method, an ICANmethod and the like, and further, a method for amplifying a gene otherthan a PCR method.

D. 4^(th) Invention

This invention is a method for determining accurately a target nucleicacid using a measuring value obtained by the methods of the presentinvention. The details will be described in Examples 8 and 11.

EXAMPLES

In this examples, various nucleic acid probes, internal standard nucleicacids, and target nucleic acids are used as an oligodeoxynucleotide,unless otherwise specifically indicated.

In various nucleic acid probes, an fluorescence-labeled portion orposition is the 5′-OH group of a sugar of the 5′-end in regard to the5′-end (the group is obtainable by dephosphorylation); on the otherhand, the 3′-OH group of a sugar of the 3′-end in regard to the 3′-end:

Example 1

A novel mixture comprising two QProbes and a novel method for assaying agene making use thereof.

Novel Mixture

The novel mixture having the following composition was prepared.

-   -   The following QProbe A (a QProbe for detecting a target gene):        200 nM;    -   the following QProbe B (for detecting a internal standard gene):        200 nM;    -   the following internal standard nucleic acid: the ones    -   having each of the following concentrations were prepared:        a) 1/10, 1/5, 1/2, 4/5, and 9/10 of 200 nM        b) 1/10, 1/5, 1/2, 4/5, and 9/10 of 800 nM;    -   Buffer composition: 10 mM Tris-HCl buffer (pH: 8.3), KCl: 50 mM,        MgCl₂: 1.5 mM

Novel Assaying Method

(1) Experimental Method

It is examined whether or not a ratio between genes existing a system iscapable of being determined using the above mixture added with a targetgene and using two QProbes. In the experiment, after DNA solutions withvaried ratios between a target gene and an internal standard gene had beprepared, and therein added with the QProbe for detecting a target geneand the QProbe for detecting an internal standard gene, a fluorescentmeasurement was implemented. The fluorescent measurement was conductedat 60° C. and 95° C. (the measuring values at 60° C. were those onhybridization of the probe with a target gene; the measuring values at95° C. were those on complete cleavage of the hybrid complex of theprobe and the target gene). Incidentally, the measuring values at 95° C.were taken as a reference so as to determine a fluorescence-quenchingrate. In addition, a relation of a ratio of the fluorescence-quenchingrates of the each probes and a existing ratio of the target gene and theinternal standard gene was examined; it was examined whether or not agene existing ratio could be determined based this relative equation.

Detailed experimental conditions were indicated in the following.

(2) Experimental Conditions

Target Gene and Internal Standard Gene

Target gene and internal standard gene: oligonucleotides were used.

Synthesis of oligonucleotide: by relying custom synthesis services(Espec oligoservices Inc.). Base sequence of target gene: 5′-AGTTC CGGAAAGGCC AGAGG AG-3′ Base sequence of internal standard gene: 5′-GGTTCCGGAA AGGCC AGAGG AA-3′

Total final concentration as combined of internal standard gene andtarget gene: 200 nM, 800 nM (target:internal standard=9:1, 4:1, 1:1,1:4, 1:9).

QProbe

Synthesis: by relying custom synthesis services (Espec oligoservicesInc.). Sequence: 5′-CTCCT CTGGC CTTTC CGGAA CC-3′

Dye: the QProbe A (a Qprobe for detecting a target gene) was influorescence labeled with BODIPY FL (Molecular probes Inc., D-6140); theQProbe B (a QProbe for detecting an internal standard gene) was labeledwith TAMRA (Molecular probes Inc., C-6123).

Final concentration of each QProbe: 200 nM (as a total concentration:400 nM)

Buffer: 10 mM Tris-HCl buffer (pH: 8.3), KCl: 50 mM, MgCl₂: 1.5 mM.

Used apparatus: fluorescence measurements were carried out by afluorometer, LS50B (PerkinElmer Inc.) with an temperature-controllinginstrument (Inc.); the measurements were conducted at 480 nm excitationwavelength and 520 nm fluorescence wavelength for the QProbe A, and at550 nm excitation wavelength and 580 nm fluorescence wavelength for theQProbe B.

Fluorescence measurements on hybridization were conducted 60° C. Afluorescent intensity at the temperature (95° C.) as a reference, thetemperature completely de-hybridizing from a probe; fluorescentquenching rates were determined based on the following equation.

A fluorescent quenching rate (%)=(1−(F60/F95)/(only F60 probe/only F95probe))×100, wherein

F60: a fluorescent intensity value at 60° C. in presence of a targetgene;

F95: a fluorescent intensity value at 95° C. in presence of a targetgene;

Only F60: a fluorescent intensity value at 60° C. in absence of a targetgene

Only F95: a fluorescent intensity value at 95° C. in absence of a targetgene.

(3) Experimental Results and Discussion

FIG. 5 illustrates the experimental results. On the basis of the graphs,it was cleared that a constituent ratio of a target gene/an internalstandard gene was highly correlative to a ratio of QProbe A quenchingrate/a QProbe B quenching rate. This method was, thereby, suggested toenable a target gene to be assayed.

Even when the total sum amount of a target gene and an internal standardgene was more than the additive amount of a probe (800 nM), the aboveco-relation was observed also like when less (200 nM) than the additiveamount; it is suggested that this method could be determined accuratelyand quantitatively. Based on the above findings, it is suggested thatthis method is a method having features such that (1) a processing stepfor diluting a gene is not necessary; and (2) it is not necessary tomake a concentration of a probe vary.

Example 2

2-2 Screening of Novel Dye Capable of Quenching Fluorescence Due to itsCo-Reaction with a Guanine.

(1) Experimental Method

A probe was prepared in which said probe was labeled at a C of a sugarof the 3′-end with a dye to be screened. A fluorescence-quenching ratewas assayed by making the prepared probe hybridize with a correspondingchain in a solution.

Experimental Procedures

(2) Experimental Conditions

Target Gene

Target gene: an oligo DNA was used.

Synthesis of oligonucleotide: by relying custom synthesis services(Espec oligoservices Inc.). Base sequence of target 3′ggggg ggggg ggAAAAAA5′ gene:

Base sequence of internal standard gene:

Final concentration: 320 nM.

Probe

Synthesis of oligonucleotide: by relying custom synthesis services(Espec oligoservices Inc.). Base sequence: 5′CCCCC CCCCC CCTTT TTT3′Dye: PacificBlue (P-10163), TET (C-6166), TBSF (C-6166), HEX (T-20091),and R6G (C-6127) were tested.

Final concentration: 40 nM.

Buffer: 10 mM Tris-HCl buffer (pH: 8.3), KCl: 50 mM, MgCl₂: 1.5 mM.

Used Apparatus

Fluorescent measurements were conducted by a fluorometer, LS50B(PerkinElmer Inc.) with a temperature-controlling instrument(PerkinElmer Inc.). Conditions for fluorescent measurements weredetermined by measuring maximum excitation wavelengths and maximumfluorescence wavelengths of each dyes using LS50B. In order tofluorescent quenching rates, measurements were conducted by using theobtained maximum excitation wavelength and maximum fluorescentwavelength. The maximum excitation wavelengths and maximum fluorescentwavelengths of each dyes were shown the description of the below resultsection. The slit width to measure was 5 nm in both excitation andfluorescent wavelengths.

FIG. 8 indicates the experimental procedures. The fluorescentmeasurement was conducted at 35° C.

The fluorescent quenching rate was determined by the following equation.Fluorescent quenching rate(%)=(measuring value(i)−measuringvalue(ii))÷measuring value(i)×100

(3) Experimental Results and Discussion

As ascertained from the bellow Table, fluorescent quenching wasconfirmed in all dyes tested. Specifically the fluorescent quenching wasmarked in PacificBlue and R6G; the fluorescent quenching rates werehigher in those than in dyes known so far. Further, PacificBlue (itsimplement unknown), R6G (its implement unknown), BODIPY FL (itsimplement known) and TAMRA (its implement known) each were different influorescent character thereof; by using four probes labeled with theabove dyes, four genes being present in the same could be detectedconcurrently. TABLE 2 Relation between fluorescent dye and quenchingrate thereof Fluorescence - Maximum Maximum quenching excitationemission rate wavelangth wavelangth Fluorescent dye (%) (nm) (nm)Unknown PacificBlue 94.0 395 450 use (P - 10163) of dye TET 56.6 515 530(C - 6166) TBSF 86.5 520 540 (C - 6166) HEX 68.3 530 542 (T - 20091) R6G93.1 517 543 (C - 6127) Known BODIPY FL 91.8 499 522 use (D - 6140) ofdye TAMRA 89.2 547 579 (C - 6123)

Example 3

2-3 Method for Assaying a Gene Using a Switching Probe Novel Mixture

The novel mixture having the following composition was prepared.

-   -   The below Switching probe: 400 nM;    -   the following internal standard nucleic acid: the ones    -   having the following concentrations each were prepared: (a)        1/10, 1/5, 1/2, 4/5, and 9/10 of 600 nM    -   Buffer composition: 10 mM Tris-HCl buffer (pH: 8.3), KCl: 50 mM,        MgCl₂: 1.5 mM.

In addition, in order to verify the experimental data obtained by usingthe Switching probe, a novel mixture was prepared by replacing thefollowing QProbe A and QProbe B for the Switching probe in the abovereaction solution—The following QProbe A (for detecting a target gene):200 nM;

-   -   The following QProbe B (for detecting an internal standard        gene): 200 nM.

Novel Assaying Method

A target gene was assayed by adding the target gene in the above novelmixture. Further, the fluorescent quenching rates obtained by using theSwitching probe and the fluorescent quenching rates obtained by usingtwo QProbe were compared.

(1) Experimental Method

It was examined whether or not the existing ratios of target genes addedin the novel mixture could determined even using the Switching probeslike using two QProbes. In addition, the comparison between thefluorescent quenching rates obtained on making use of a Switching probeand those obtained on making use of two QProbes was conducted.

Detailed experimental conditions are shown in the following.

(2) Experimental Conditions

Target Gene and Internal Standard Gene

Target gene and internal standard gene: oligonucleotides were used.

Synthesis of oligonucleotide: by relying custom synthesis services(Espec oligoservices Inc.). Base sequence of target gene: 5′-TTTGG ATGACTGACT GACTG ACTGA CGAGA TTT-3′. Base sequence of internal standard gene:TTTAG ATGAC TGACT GACTG ACTGA CGAGG TTT-3′.

Total final concentration as combined of internal standard gene andtarget gene: 600 nM (target: internal standard=9:1, 4:1, 1:1, 1:4, 1:9).

QProbe

Synthesis: by relying custom synthesis services (Espec oligoservicesInc.). Base sequence and structure of Switching probe BODIPY FL-5′CCTACTGACT GACTG ACTGA CTGCT CC3′- TAMRA Base sequence and structure ofQProbe <QProbe A (for detecting a target gene) BODIPY FL-5′CCTAC TGACTGACTG ACTGA CTGCT CC3′ <QProbe B (for detecting an internal gene)5′CCTAC TGACT GACTG ACTGA CTGCT CC3′-TAMRA

Incidentally, used fluorescent dyes were like those of Example 1.

Final concentration: 400 nM (400 nM as a total sum in making use of twoQProbes)

Buffer: 10 mM Tris-HCl buffer (pH: 8.3), KCl: 50 mM, MgCl₂: 1.5 mM.

Used apparatus, used instrument, assaying conditions, and method fordetermining a fluorescent quenching rate were like those of Example 2-1

(3) Experimental Results and Discussion

BODIPY FL was labeled on C of the 5′-end; the hybridization with atarget gene resulted in a marked fluorescent quenching. On the otherhand, TAMRA was labeled on C of the 3′-end; the hybridization with aninternal standard gene resulted in a marked fluorescent quenching.Thereby, a ratio between the quenching rate for BODIPY FL and thequenching rate for TAMRA was expected to be highly co-relative to aexisting ratio of a target gene and an internal standard gene. FIG. 6illustrates the experimental results. It is ascertained that a ratiobetween the quenching rate for BODIPY FL and the quenching rate forTAMRA is highly co-relative to an existing ratio of a target gene and aninternal standard gene. These results indicate that a target gene iscapable of being determined quantitatively even by making use of aSwitching probe.

The fluorescent quenching rates obtained on making of the Switchingprobe and the fluorescent quenching rates obtained on making use of twoQProbe were compared. Those results are shown in Table 3. As confirmedfrom this table also, the fluorescent quenching rates obtained on makinguse of Switching probe is about two-times higher than those obtained onmaking use of two QProbes. This fact indicates that the use of aSwitching probe enables more accurate determination of a target genethan the use of two QProbes. TABLE 3 Comparison between quenching rateon use of Switching probe and that on use of two QProbes Conditions foruse Concen- Concen- tration tration of Probes of target Results (nM)Target (nM) Switching BODIPY FL 35.2% 400 Target 600 probe Quenchinggene rate TAMRA 72.5% 400 Internal 600 Quenching standard rate gene TwoQProbe A 21.2% 400 Target 600 QProbes Quenching gene rate QProbe B 44.2%400 Internal 600 Quenching standard rate gene

Example 4

2-4 Method for Assaying a Target Gene at an End Point Through aGene-Amplifying Method

(1) Experimental Method

It was tried to ascertain whether or not a target gene could be assayedprecisely by the method for assaying a target gene at an end point. APCR method was adopted as a gene-amplifying method; and a Switchingprobe was adopted a probe.

(2) Experimental Conditions

Target Gene and Internal Standard Gene

Target gene: a PCR product of a soybean (round-up soybean) recombinantRRS gene; the base sequence of the target gene: 5′-AGTTC CGGAA AGGCCAGAGG AG-3′

(the underlined portions are different from those of an internalstandard gene. The probe can hybridize with the described sequence. Thesequence other than those is common to that of the target gene orinternal standard gene; thereby, it is not described.).

Internal standard gene: a PCR product of a soybean (round-up soybean)recombinant RRS gene artificially incorporated with a mutation. Basesequence of the internal standard gene: 5′-GGTTC CGGAA AGGCC AGAGG AA-3′

(The underlined portion is different from that of the target gene. Theprobe was capable of hybridizing with the described sequence. Thesequence other than those was common to that of the target gene orinternal standard gene; thereby, it is not described.).

Total final concentration as combined of internal standard gene andtarget gene: 1000 copy

Starting constituent ratio: target: internal standard=9:1, 4:1, 1:1,1:4, 1:9.

Preparation of Switching Probe

Synthesis: by relying custom synthesis services (Espec oligoservicesInc.).

Base sequence and structure of Switching probe BODIPY FL-5′CTCCT CTGGCCTTTC CGGAA CC3′-TAMRA

(Dyes are like the above)

Final concentration of probe: 400 nM

Conditions of PCR

Denaturing: 95° C. (60 seconds)

Annealing: 56° C. (60 seconds)

Extension: 72° C. (60 seconds)

-   -   Enzyme for amplification of gene: Gene Taq (Nihon Gene Inc.) was        used as a Taq polymerase

Primer

Synthesis: by relying custom synthesis services (Espec oligoservicesInc.). Base sequence of 5′ CCTTT AGGAT TTCAG CATCA GTGG3′ forwardprimer: Base sequence of 5′ GACTT GTCGC CGGGA ATG3′ reverse primer:

Final concentration of primer: each 1000 nM

Preparation of Internal Standard Gene

An oligoDNA having a base sequence of a internal standard gene astargeted was prepared by relying custom synthesis services (Especoligoservices Inc.). The obtained oligoDNA was amplified, using itselfas a temperate, by a PCR amplification method using the above primer fordetecting; the amplification product was purified using Microcon PCR(Millipore Inc.). Following the purification, the amplification productwas assayed quantitatively using PicoGreen (Molecular probes Inc.); theamplification product obtained in such a way is used as an internalstandard gene.

Preparation of PCR Product of Target Gene

It was prepared like the preparation method of the internal standardgene.

Used Apparatus and Measuring Conditions

Used apparatus, used instrument, assaying conditions, and method fordetermining a fluorescent quenching rate on determination of fluorescentquenching rates were like those of Example 2-1

Quantitative determination using PicoGreen was conducted at 480 nm as anexcitation wavelength and 520 nm as a fluorescent wavelength. Used slitwidth was 5 nm in any wavelengths. The PCR was conducted using a thermalcycler (iCycler, Biorad Inc.).

(3) Experimental Results

It was ascertained that a ratio between a fluorescent quenching rate forBODIPY FL and a fluorescent quenching rate for TAMRA was highlyco-relative to the existing ratio between a target gene and an internalstandard gene based on the obtained relative equation between those.From these results, it was suggested that a target gene and an internalstandard gene was amplified with keeping a starting gene-constituentratio. In addition, this method was shown to be capable of assaying atarget gene through a gene amplification method.

Example 5

In regard to a novel mixture comprising a nucleic acid probe for ahomogenous solution system and an internal standard nucleic acid and amethod for assaying a gene by use thereof, preferable examples are shownin the following.

(1) Experimental Method

A possibility was examined of determining an existing ratio of a targetgene and internal standard gene by making use of a probe for ahomogenous solution system (a QProbe was used here). In the experiment,after preparation of a DNA solution containing a target gene and aninternal standard gene with varied existing ratios thereof, a QProbe wasadded in the DNA solution and then fluorescent measurements wereconducted. The fluorescent measurements were conducted at 60° C. and 95°C. (the measuring values at 60° C. were those on hybridization of theprobe with a target gene; the measuring values at 95° C. were those oncomplete cleavage of the hybrid complex of the probe and the targetgene). Incidentally, the measuring values at 95° C. were taken as areference so as to determine a fluorescence-quenching rate. In addition,a relation of a ratio of the fluorescence-quenching rates of the eachprobes and a existing ratio of the target gene and the internal standardgene was examined; it was examined whether or not a gene existing ratiocould be determined based this relative equation.

Detailed experimental conditions are shown below.

(2) Experimental Conditions

Target Gene and Internal Standard Gene

Target gene and internal standard gene: oligonucleotides were used.

Synthesis of oligonucleotide: by relying custom synthesis services(Espec oligoservices Inc.). Base sequence of target gene: 5′-AGTTC CGGAAAGGCC AGAGG AG-3′ Base sequence of internal standard gene: 5′-GGTTCCGGAA AGGCC AGAGG AA-3′

Total final concentration as combined of internal standard gene andtarget gene: 200 nM, 400 nM, 800 nM (target:internal standard=9:1, 3:1,1:1, 1:3, 1:9).

QProbe

Synthesis: by relying custom synthesis services (Espec oligoservicesInc.). Sequence: 5′-CTCCT CTGGC CTTTC CGGAA CC-3′

Dye: BODIPY FL (Molecular probes Inc., D-6140).

Final concentration of each QProbe: 400 nM

Buffer: 10 mM Tris-HCl buffer (pH: 8.3), KCl: 50 mM, MgCl₂: 1.5 mM.

Used apparatus: fluorescence measurements were carried out by afluorometer, LS50B (PerkinElmer Inc.) with a temperature-controllinginstrument (Inc.); the measurements were conducted at 480 nm excitationwavelength and 520 nm fluorescence wavelength.

Fluorescence measurements on hybridization were conducted 60° C. Afluorescent intensity at the temperature (95° C.) as a reference, thetemperature completely dehybridizing from a probe; fluorescent quenchingrates were determined based on the following equation.A fluorescent quenching rate(%)=(1-(F60/F95)/(only F60 probe/onlyF95))×100, wherein

F60: a fluorescent intensity value at 60° C. in presence of a targetgene;

F95: a fluorescent intensity value at 95° C. in presence of a targetgene;

Only F60: a fluorescent intensity value at 60° C. in absence of a targetgene

Only F95: a fluorescent intensity value at 95° C. in absence of a targetgene.

(3) Experimental Results and Discussion

FIG. 10 shows the experimental results. When the total sum amount of thetarget gene and the internal standard gene was equal to the additiveamount of the probe (400 nM), and when more than the additive amount ofthe probe (800 nM), the calibration curves agreed with each other. Thisshowed that, when the amount of the probe was not more than the totalsum of the amount of a gene, the gene-constituent ratio obtained ondetermination of a fluorescent quenching rate for the BODIPY dye wasequaled regardless of a total sum of the amount of a gene. Accordingly,it is shown that, when the amount of a probe is not more than a totalsum of the amount of a gene, a gene-constituent ratio can be accuratelydetermined quantitatively based on an obtained fluorescent quenchingrate for a BODIPY dye. The calibration curve in the case that the amountof a probe was more than the total sum of the amount of the gene (200nM), however, was not agreed with the calibration curve in the case thatthe amount of the probe was not more than the total sum of the amount ofthe gene. This was interpreted by an because that, when a total sum ofthe amount of the gene was 200 nM, the additive amount of the probe was400 nM; the non-hybridized probe was present 200 nM; thereby afluorescent quenching rate was rendered about a half as compared withthat in the case when the total sum of the amount of the gene was 400 nMor 800 nM.

Incidentally, in the case when a total sum of the amount of a gene (aconcentration of a target nucleic acid) was not more than the amount ofa probe, a measurement was preferable to conduct with a probe making itsamount vary in order to solve a problem of the non-agreeable calibrationcurve.

Example 6

Method for Assaying a Gene Using a Novel Mixture Comprising aDoubly-Labeled Nucleic Acid Probe and Internal Standard Nucleic Acid(Part 2)

Although, in the method as described in Example 5 also, a target nucleicacid is capable of being assayed accurately by making the amount of aprobe vary variously, this method can preferably assay a target nucleicacid using a probe making its amount vary variously.

(1) Experimental Method

It was examined whether or not a existing ratio of target nucleic acidswas capable of being assayed like the case of making use of theabove-described doubly-labeled probe (a probe labeled at C or G self) byusing a doubly-labeled probe of such a type that the probe was renderedvariable in the fluorescent intensity by causing the number of G of atarget nucleic acid and internal standard nucleic acid to change betweenthe both in a range of from the 1^(st) base to the 3^(rd) from the basecorresponding to a fluorescence-labeled portion (a base corresponding toa labeled base being counted as the 1^(st) base), wherein thedoubly-labeled probe was completely complementary to the target nucleicacid and internal standard nucleic acid (FIG. 11).

Detailed experimental conditions is described in the following.

(2) Experimental Conditions

Target Gene and Internal Standard Gene

Target gene and internal standard gene: oligonucleotides were used.

Synthesis of oligonucleotide: by relying custom synthesis services(Espec oligoservices Inc.).

Base sequence of target gene (a sequence underlined is capable ofhybridizing to the probe) 5′-TAATG ATGAC TGACT GACTC ACTGA CGATG CT-3′

Base sequence of internal standard gene (a sequence underlined iscapable of hybridizing to the probe)5′-TGGTA TGACT GACTG ACTGA CTGAC GAGTA AT-3′

Total final concentration as combined of internal standard gene andtarget gene: 200 nM, 400 nM, 800 nM (target:internal standard=9:1, 3:1,1:1, 1:3, 1:9).

Doubly-Labeled Nucleic Acid Probe BODIPY FL-5′ACTAC TGACT GACTG ACTGACTGCT CA 3′- TAMRA

Final concentration: 400 nM

Incidentally, used fluorescent dyes and relying custom synthesisservices and the like were like Example 5.

-   -   Used apparatus, used instrument, conditions for assaying, and        method for determining a fluorescent quenching rate were like        those of Example 5.

Marked fluorescent quenching for BODIPY FL of the doubly-labeled probewas caused because of the presence of G in near proximity of afluorescence-labeled end base on its hybridization to the target gene;but the hybridization of the doubly-labeled probe to the internalstandard nucleic acid led to no fluorescent quenching because of theabsence of G in near proximity of a fluorescence-labeled end base.

On the other hand, for BODIPY FL of the doubly-labeled probe, markedfluorescent quenching for was caused because of the presence of G innear proximity of a fluorescence-labeled end base on hybridization ofthe doubly-labeled probe to the internal standard nucleic acid.Therefore, a ratio between a fluorescent quenching rate for BODIDY FLand a fluorescent quenching rate for TAMRA was expected to be highco-relative to a ratio between a target gene and internal standard gene.

(3) Experimental Results and Discussion

The experimental results are shown in FIG. 12. From the graphs, a ratiobetween a fluorescent quenching rate for BODIDY FL and a fluorescentquenching rate for TAMRA is ascertained to be high co-relative to aratio between a target gene and internal standard gene. It is suggestedfrom these results that the method using the doubly-labeled nucleic acidprobe of this model is capable of accurately assaying a target genequantitatively in any case where the total amount of a gene was largerthan that of a probe, or smaller.

Example 7

Method for Assaying a Gene Using a Novel Mixture Comprising aDoubly-Labeled Nucleic Acid Probe and Internal Standard Nucleic Acid(Part 3)

(1) Experimental Method

It was examined whether or not an existing ratio of target nucleic acidswas capable of being determined, like using the above describeddoubly-labeled probe, using also a doubly-labeled nucleic acid probedesigned such that, in regard to a fluorescent dye labeled to oneportion of two labeled portions of the a doubly-labeled nucleic acidprobe, the change amount in a fluorescent character was caused in asimilar degree on hybridization of the one portion to any of a targetnucleic acid and an internal standard nucleic acid; but, in regard to afluorescent dye labeled to the other portion, a difference between thechange amount in a fluorescent character on hybridization of the otherportion with the target nucleic acid and the change amount in afluorescent character on hybridization of the other portion with theinternal standard nucleic acid was caused (FIG. 13).

Detailed experimental conditions are described in the following.

(2) Experimental Conditions

Target Gene and Internal Standard Gene

Target gene and internal standard gene: oligonucleotides were used.

Synthesis of oligonucleotide: by relying custom synthesis services(Espec oligoservices Inc.).

Base sequence of target gene (a sequence underlined is capable ofhybridizing to the probe) 5′-TAAGG ATGAC TGACT GACTG ACTGA CGATG GT-3′

Base sequence of internal standard gene (a sequence underlined iscapable of hybridizing to the probe)5′-TAAGA TGACT GACTG ACTGA CTGAC GAGTA AT-3′

Total final concentration as combined of internal standard gene andtarget gene: 200 nM, 400 nM, 800 nM (target:internal standard=9:1, 3:1,1:1, 1:3, 1:9).

Base Sequence and Structure of Doubly-Labeled Nucleic Acid Probe BODIPYFL-5′ACTAC TGACT GACTG ACTGA CTGCT CC 3′- TAMRA

Final concentration: 400 nM

Incidentally, used fluorescent dyes were like Example 5.

-   -   Used apparatus, used instrument, conditions for assaying, method        for determining a fluorescent quenching rate, buffer and the        like were like those of Example 5.

Marked fluorescent quenching for BODIPY FL labeled at the 5′-end of thedoubly-labeled probe was caused because of the presence of nearproximity of a fluorescence-labeled end base on its hybridization to thetarget gene; but the hybridization of the doubly-labeled probe to theinternal standard nucleic acid led to no fluorescent quenching becauseof the absence of G in near proximity of a fluorescence-labeled endbase. Thus, it is thought that a fluorescent quenching rate is in highcorrelation of an existing ratio of target nucleic acids. Further, if aproportion in quantity of a hybridized probe is known, the proportion ofa probe hybridized to a target nucleic acid (or an internal standardnucleic acid) to the total amount of the hybridized probe comes to bepossible to determine. This will be explained based on the followingexamples. In the case that it was calculated that the quenching rate fora fluorescent dye having a difference in a fluorescent quenching levelcaused on hybridization between a target nucleic acid and an internalstandard nucleic acid in a practical sample was 40%; and the half of thetotal amount of an added probe was hybridized, if on paying attention toonly a hybridized probe, the 80% amount of the hybridized probe comes tocause a fluorescent quenching. Provided that a probe used herein istaken to be a probe causing to quench completely a fluorescent emissionof a BODIPY FL dye on hybridization of the probe to a target gene, the80% amount of the hybridized probe is capable of being calculated basedon the quenching rate (40%) in the practical sample. Thus determinationof a proportion in quantity of a hybridized probe and afluorescence-quenching made it possible to determine quantitatively aconstituent ratio between a target gene and internal standard gene.

Fluorescent quenching for TAMRA labeled at the 3′-end of thedoubly-labeled probe was markedly caused in similar levels onhybridization to a target gene and on hybridization to an internalstandard gene because of the presence of G in near proximity of afluorescence-labeled end base in the both of the target gene and theinternal standard gene on its hybridization to the target gene. Becauseof this, the quenching rate for a TAMRA dye came to indicate aproportion of a hybridized probe.

As described above, it is thought that, if a proportion of a hybridizedprobe is known, a constituent ratio of a gene is capable of bedetermined quantitatively. A target gene was capable of be determinedquantitatively by correction of a quenching rate for a BODIPY FL dyewith a quenching rate for a TAMRA dye irrespective of a magnitude of thetotal amount of a gene.

(3) Experimental Results and Discussion

FIG. 14 shows experimental results. FIG. 14A shows a correlation of afluorescence-quenching rate for a BODIPY dye and an existing ratio of atarget gene. From this figure, it was understood that calibration curveseach other was different with depending on the total amount of a targetgene. These results indicate that a fluorescence-quenching varies withchanged amounts of genes even if existing ratios of target genes are thesame. From this, it is apparently impossible to determine an existingratio of target genes. On the other hand, in a value (afluorescence-quenching rate for a BODIPY dye/a fluorescence-quenchingrate for a TAMRA dye) obtained by dividing a fluorescence-quenching ratefor a BODIPY dye by a fluorescence-quenching rate for a TAMRA dyeindicating a proportion in quantity of a hybridized probe, it isunderstood that these equations were almost in agreement irrespective ofdifferent amounts of a gene (FIG. 14B). In conclusion, a value of afluorescence-quenching rate for a BODIPY dye/a fluorescence-quenchingrate for a TAMRA dye was confirmed to change with depending on only aexisting ratio of target genes and without depending on the total amountof a target gene. It is suggested from the above results that the methodusing a doubly-labeled probe of the above type is capable of determiningan existing ratio of target genes accurately and quantitativelyirrespective of a magnitude of the total amount of a target gene.

Insofar as that a probe usable in the method can have features such asfollows: (1) the fluorescence amount of a dye labeled at one portion ofthe probe on hybridization of the probe with a target gene is differentfrom that on hybridization of probe with an internal standard gene, and(2) in a dye labeled at another portion, the fluorescence amount (it maybe a fluorescence-quenching or a fluorescence-emission) is caused insimilar levels on hybridization with either a target gene or an internalstandard gene, namely insofar as the probe has such properties, thismethod is capable of assaying any gene based on the above principal.Insofar as that a change in a fluorescent character in a dye usable forthis method may be caused on hybridization of a probe with a gene, anydye may be usable, namely insofar as a dye has such properties,specifically no limitation is imposed on a dye.

Example 8

Method for Accurately Calculating Concentration of Target Nucleic AcidBased on Fluorescence Measuring Value

Probes used in the above Examples (excluding Example 7) were probes suchthat fluorescence amounts for dyes with which the probes were labeledvaried on hybridization of the probes with any one of a target gene andinternal standard gene; on hybridization of the probes with the othergene, the fluorescence amounts were in similar levels as compared withthat on no hybridization. That is, the existing forms of a probe werethree forms wherein one of those is a target gene and probe hybridizingcomplex form, the other an internal standard gene and probe hybridizingcomplex form, and the further one a non-hybridizing form; but, upon thepayment of attention to the fluorescence amount for every one moleculeof a dye, the existing forms of a probe wherein the forms each weredifferent in the fluorescence amount were only two forms (see FIG. 15).However, a probe has been recognized to be present during the aboveachievement of the invention, which probe had three existing forms whicheach were different in the fluorescence amount, wherein the forms werethree forms of a target gene and probe hybridizing complex form, aninternal standard gene and probe hybridizing complex form, and anon-hybridizing form (see FIG. 15). Because there were a probe havingthree existing forms different in the fluorescence amount in a reactionsystem using a probe of such above type, it was thought to be difficultto determine a target nucleic acid by making use of a simplecalculating-equation as described above. Accordingly, a calculatingmethod for determining a target gene quantitatively was invented, whichmethod was designed on the premise of the use of a probe wherein thefluorescence amount for the probe was different independently in atarget gene and probe hybridizing complex form, an internal standardgene and probe hybridizing complex form, and a non-hybridizing form; itwill be illustrated in the following description.

<Calculating Equation>

A calculating equation is illustrated in the following description,which equation is usable in the case using a doubly-labeled probedesigned such that the fluorescent intensity for a dye with which aprobe is labeled at one portion thereof is reduced remarkably onhybridization with a target gene; that for a dye with which the probewas labeled at the other portion thereof was reduced remarkably onhybridization with an internal standard gene.

That detail is described as follows.

Signs usable in the calculating equation are defined as follows:

y: a proportion of a hybridized probe;

1−y: a proportion of a non-hybridized probe;

x: a proportion of a target gene;

1−x: a proportion of an internal standard gene;

A: a ratio of the fluorescent intensity of dye A of the doubly-labelednucleic acid probe on no hybridization in use of a practical sample tothe fluorescent intensity of dye A of the doubly-labeled nucleic probeon no hybridization;

a: a ratio of the fluorescent intensity of dye A of the doubly-labelednucleic acid probe on its 100%-hybridization with a target nucleic acidto the fluorescent intensity of dye A of the doubly-labeled nucleic acidprobe on no hybridization;

a′: a ratio of the fluorescent intensity of dye A of the doubly-labelednucleic acid probe on its 100%-hybridization with an internal standardnucleic acid to the fluorescent intensity of dye A of the doubly-labelednucleic acid probe on no hybridization;

B: a ratio of the fluorescent intensity of dye B of the doubly-labelednucleic acid probe on hybridization in use of a practical sample to thefluorescent intensity of dye B of the doubly-labeled nucleic probe on nohybridization;

b: a ratio of the fluorescent intensity of dye B of the doubly-labelednucleic acid probe on its 100%-hybridization with a target gene to thefluorescent intensity of dye B of the doubly-labeled nucleic acid probeon no hybridization;

b′: a ratio of the fluorescent intensity of dye B of the doubly-labelednucleic acid probe on its 100%-hybridization with an internal standardnucleic acid to the fluorescent intensity of dye B of the doubly-labelednucleic acid probe on no hybridization.

The fluorescent intensity proportion (A) of dye A in use of a practicalsample is capable of being represented as follows:A=(1−y)+axy+(1−x)a′y=1−y+axy+a′y−a′xy  (1)

If the equation (a) is deformed, a proportion (y) of a hybridized probeis capable of being represented as follows:y=(1−A)/(a′x−ax+1−a′)  (2)

On the other hand, a fluorescent intensity (B) for dye B in use of apractical sample is capable of being represented.B=(1−y)+(1−x)by +b′xy=1−y−bxy+by +b′xy  (3)

If the equation (3) is deformed, a proportion of a hybridized probe iscapable of being represented as follows:y=(1−B)/(b′x−bx+1−b′)  (4)

The equation (2) is incorporated with the equation (4); the resultantequation is represented as follows:(1−A)/(a′x−ax+1−a′)=(1−B)/(b′x−bx+1−b′)  (5)

The deformation of the equation (5) leads the equation (6):X=(−a′−B+Ba′+b′+A−Ab′)/(b′−b−Ab′+Ab−a′+a+Ba′−Ba)  (6)

The items other than x are determined experimentally; a proportion of atarget gene is thought to be capable of being calculated by an equation(6).

Detailed experimental conditions will be described below.

In the experiment, a constituent ratio of target genes in samplesprepared imitatively (hereinafter, called simply a “mimic sample”.) wasdetermined by (1) a method comprising, subsequent to preparation of acalibration curve, determining quantitatively a constituent ratio oftarget genes by the prepared calibrating curve; or (2) a methodcomprising determining quantitatively a constituent ratio of targetgenes by the above calculating equation; and, based on the obtainedresults, the above two methods for determining quantitatively aconstituent ratio of target genes were compared and evaluated.

(2) Experimental Conditions

<Target Gene and Internal Standard Gene>

Target gene and internal standard gene: an oligonucleotide was used.

Synthesis of oligonucleotide: by relying custom synthesis services(Espec oligoservices Inc.).

Base sequence of target gene (a sequence underlined is capable ofhybridizing to the probe) 5′-AATTC GTACC AACTA TCCTC GTCGT CAGCT ATG-3′

Base sequence of internal standard gene (a sequence underlined iscapable of hybridizing to the probe)5′-GATTC GTACC AACTA TCCTC GTCGT CAGCT ATA-3′

Additive amount of the target gene and internal standard gene in themeasurement of the fluorescent quenching rate

Target gene: 800 nM

Internal standard gene: 800 nM

Total final concentration as combined of internal standard gene andtarget gene (analytical curve): 800 nM (target: internal standard=9:1,3:1, 1:1, 1:3, 1:9).

Total final concentration as combined of internal standard gene andtarget gene (mimic sample): 200 nM, 800 nM (target: internal standardgene=4:1).

Base Sequence and Structure of Doubly-Labeled Nucleic Acid Probe BODIPYFL- 5′CATAG CTGAC GACGA GGATA GTTGG TACGA ATC 3′-TAMRA

Incidentally, used fluorescent dyes were like Example 6. Finalconcentration of the probe was 400 nM.

The other conditions (used apparatus, used instrument, conditions forassaying, and the like) were like those of Example 7.

(3) Results and Discussion

<Measurement of Fluorescence-Quenching Rate of Probe>

Firstly, the fluorescence-quenching rate—in a target gene and probehybridizing complex form, an internal standard gene and probehybridizing complex form, and a non-hybridizing form—of the probe usedin this example were measured. Incidentally, in the measurement of thefluorescent quenching rate for a BODIPY FL dye, a fluorescent quenchingrate obtained in an experimental system where an internal standard genewas added therein was used as a basal unit (the quenching rate=0%); theother quenching rates were calculated based on the basal unit. In themeasurement of the fluorescent quenching rate for a TAMRA dye, afluorescent quenching rate obtained in an experimental system where atarget gene was added therein was used as a basal unit (the quenchingrate=0%); the other quenching rates were calculated based on the basalunit. The results are shown in the following table. As understood fromthe table, the fluorescent quenching rates of an experimental systemwith no addition of a gene (a gene-non-additive system) were not 0% forboth dyes. These findings indicate that, on hybridization of the probewith a target gene also—the gene not having a G in the base-sequenceposition complementary to a labeled base of the probe, the fluorescenceamount emitted from the dye labeled to the probe was variable1. From theabove findings, it is ascertained that the fluorescence amount for theprobe used in this example varies in a target gene and probe hybridizingcomplex form, an internal standard gene and probe hybridizing complexform, or a non-hybridizing form. TABLE 4 Quenching rate in eachconditions of probe used in Example BODIPY FL TAMRA Addition AdditionAddition of only Addition of only No of only internal No of onlyinternal addition target standard addition target standard of gene geneof gene gene gene (800 nM) (800 nM) gene (800 nM) (800 nM) Fluorescent110 122 105 87 82 91 intensity (95° C.) Fluorescent 129 24 117 135 14520 intensity (60° C.) 60° C./95° C. 1.17 0.19 1.11 1.56 1.77 0.22Quenching −5.0 82.6 0.0 12.0 0.0 87.5 rate (%)

<Determination of Existing Ratio of Target Gene>

The below figure illustrates a calibrating curve. It is recognized thatan existing ratio of a target gene is highly co-relative to afluorescence-quenching rate. Accordingly, existing ratios of targetgenes in mimic samples were determined quantitatively based on therelative equation obtained from this calibrating curve.

The results of the determination are shown in the below table. Fromthese results, it is understood that appropriate determining values cometo having been obtained as determining values in the case that the totalsum amount of a target gene was 800 nM, because the determining valueswere close to the theoretical value (a target gene: an internal standardgene in the mimic samples were 4:1; the theoretical value was 4.0). Whenthe total sum amount of genes was, however, 200 nM, the obtainedconstituent ratio of the target genes was about half of the theoreticalvalue. Based on the above results, it is suggested that a constituentratio of genes are capable of being appropriately determined in the casethat the amount of a probe is smaller than the total sum amount ofgenes, and in the case that the determination was conducted using aprobe designed such that the fluorescence amount for the probe used inthe determination was different in no-hybridization with any gene, inhybridization with a target gene and in hybridization with an internalstandard gene, respectively. TABLE 5 Quantitative ratio of target genesexisting in a system (target gene/internal standard gene), obtained byuse of calibrating curve Total BODIPY TAMRA BODIPY - QuenchingQuantitative value Theoritical value amount FL Quenching Quenchingrate/TAMRA - (target/internal (target/internal of gene rate rateQuenching rate standard) standard) Mimic 63.4 17.3 3.66 3.82 4.0 sample1 (target:internal (※ 1) standard = Mimic 31.2 14.6 2.13 2.22 4:1)sample 2 (※ 2)Added amount of probe: 400 nM※ 1; Total amount of probe: 200 nM※ 2; Total amount of probe: 800 nM

The additive amount of a probe in this example was 400 nM; in the casewith the total sum amount of genes being 200 nM, the half of theadditive amount of the probe does not hybridize to genes. In the probeused this example, the fluorescence amount for a dye labeled at theprobe was different in no hybridization with any genes, in hybridizationwith a target gene and in hybridization with an internal standard gene,respectively; in the case when the total sum amount of genes is smallerthan the amount of a probe, the change amount in the fluorescence foreach dye labeled at the probe comes to be derived from the both of thechange amount in the fluorescence on hybridization with an internalstandard gene and the change amount in the fluorescence on hybridizationwith a target gene (in the case when the change amount in thefluorescence on no hybridization with any genes was taken as a basalvalue). That is, the change amount in the fluorescence in regard to eachdye came to be affected by the both of a target gene and an internalstandard gene. Because of such a situation, it is understood that aratio of change amounts in fluorescence did not reflect a ratio of aconstituent ratio of genes; and appropriate determining values were notcapable of being obtained. On the other hand, in the case of the totalsum amount of genes of 800 nM, a probe hybridized to any of a targetgene and an internal standard; a probe not hybridized was not present.Thereby, the change amount in fluorescence was derived from only onegene of the target gene and the internal standard gene. Accordingly, aratio of the change amount in fluorescence came to reflect appropriatelyon a constituent ratio of genes; an appropriate determining value isthought to have been obtained.

The following Table 6 shows the results of determination of aconstituent ratio of genes using the above described calculatingequation. The measuring values—those values were used for determiningquantitatively by a calibrating curve—was divided by the fluorescenceamount for a non-hybridized probe; the obtained values were incorporatedin the calculating equation. Based on this table, it is recognized thatan accurate constituent ratio of genes (the amount of a target gene/theamount of an internal standard gene) had be capable of being determinedeven in the case when the total sum amount of genes was smaller than thetotal amount like in the case when larger. Therefore, a constituentratio of genes is clearly capable of being quantitatively determinedusing the above-described relative equation nevertheless of the totalsum amount of genes. TABLE 6 Quantitative gene - constituent ratio(target gene/internal standard gene), obtained by use of novelcalculating equation Quantitative Theoritical BODIPY TAMRA value valueRelative Relative (target/ (target/ fluorescent fluorescent internalinternal intensity intensity standard) standard) Non - 1.00 1.00 — —hybridizing probe 100% target 0.17 1.14 — — gene (BODIPY: a, TAMRA: b)100% internal 0.95 0.14 — — standard gene (BODIPY: a′, TAMRA: b′) MimicSample 1 0.66 0.97 4.02 4.00 Total amount of gene: 200 nM (BODIPY: A,TAMRA: B) Mimic Sample 2 0.35 0.94 4.00 Total amount of gene: 800 nM(BODIPY: A, TAMRA: B)

Example 9

Example determining a target gene by using novel mixture according topresent invention, comprising quenching substance or probe labeled withquenching substance.

In the case when the amount of a total sum of genes is greatly smallerthan the additive amount of a probe, it is expected that the amount of achange in fluorescence for the probe become smaller; and, as a results,a target gene could not be accurately determined quantitatively (seeFIG. 17A). If fluorescent emission from non-hybridizing probe is,however, capable of being quenched, the amount of a change influorescence could be determined; as a result, even in the case when theamount of a total sum of genes was greatly smaller than the additiveamount of a probe, a target gene was expected to be capable of beingdetermined (see FIG. 17 B). In order to achieve this object, it wasthought to be effective to make use of a probe labeled with afluorescence-quenching substance (called a “quenching substance-labeledprobe”). As conditions for preferable use of the quenchingsubstance-labeled probe, the following three requirements should bementioned: (1) the quenching substance-labeled probe can hybridize to atarget nucleic acid probe; (2) the quenching substance, with which thequenching substance-labeled probe is labeled, can be located in closeproximity to a dye, with which the target nucleic acid probe is labeled,on hybridization of the quenching substance-labeled probe with thetarget nucleic acid probe; and (3) the dissociation temperature of ahybrid complex between the target nucleic acid probe and the quenchingsubstance-labeled probe can be lower than that of a hybrid complexbetween the target nucleic acid probe and a target nucleic acid or thatof a hybrid complex between the target nucleic acid probe and aninternal standard nucleic acid. An additionally preferableapplicable-example is shown in FIG. 17B. The quenching substance-labeledprobe has the following features; (1) it has a base sequencecomplementary to that of a doubly-labeled probe; (2) on hybridization tothe doubly-labeled probe, the base of the quenching substance-labeledprobe complementary to a fluorescence-labeled base of the doubly-labeledprobe is labeled with a quenching substance; and (3) it has twooligonucleotides shorter than that of a target nucleic acid probe; andeach of the oligonucleotides can hybridize to a separate portion of thetarget nucleic acid probe without doubling.

The above features of (1), (2) and (3) can cause the fluorescence of adoubly-labeled nucleic acid probe to quench without inhibition ofhybridization of the doubly-labeled nucleic acid probe to an internalstandard nucleic acid and a target nucleic acid.

(1) Experimental Method

The experiment was conducted in a system with addition of the quenchingsubstance-labeled nucleic acid probe and in a system without addition;the effect of the quenching substance-labeled nucleic acid probe wasevaluated by comparison of determining values as to existing ratios oftarget genes in mimic samples. The determination of a existing ratio oftarget genes was carried out by making use of the calculating equationas described in Example 8.

Prior to the above experiment, ranges of specific temperature were inadvance determined; at one of the ranges the doubly-labeled nucleic acidprobe was capable of hybridizing enough to only a target gene and aninternal standard gene, but of not hybridizing to the quenchingsubstance-labeled nucleic acid probe; at the other range thedoubly-labeled nucleic acid probe was capable of hybridizing enough to atarget gene, an internal standard gene and the quenchingsubstance-labeled nucleic acid probe. The temperature range was 55 to60° C. in the former and not higher than 40° C. in the later. Based onthe results, in ascertaining experiment, the temperature was changed to95° C., 57° C., and 35° C. in this order and kept for one minute to anindividual temperature. The object of keeping the temperature to 57° C.was to make the doubly-labeled nucleic acid probe hybridize to only atarget gene and an internal standard gene. Subsequent to that, thekeeping of 35° C. made the doubly-labeled nucleic acid hybridize to thequenching substance-labeled nucleic acid probe to cause the fluorescencefor the doubly-labeled nucleic acid to be quenched.

(2) Experimental Conditions

<Target Gene, Internal Standard Gene, and Doubly-Labeled Nucleic AcidProbe>

-   -   Target gene, internal standard gene, and doubly-labeled nucleic        acid probe: the same as in Example 8 were used.    -   Quenching substance-labeled nucleic acid probe

DABCYL (Glean research Inc., USA) was used. The base sequence isdescribed as follows.

Quenching substance-labeled nucleic acid probe A: 5′CTCGT CGTCA GCTAT GG3′-DABCYL

Quenching substance-labeled nucleic acid probe B: DABCYL-5′GGATT CGTAC CAACT ATC

(a region underlined is capable of hybridizing to the doubly-labeledprobe)

Synthesis of oligonucleotide: by relying custom synthesis services(Espec oligoservices Inc.)

-   -   Total final concentration as combined of internal standard gene        and target gene in mimic sample: 20 nM, 40 nM, 80 nM (target:        internal standard=4:1)    -   Final concentration of doubly-labeled probe: 400 nM    -   Final concentration of quenching substance-labeled probe: 800 nM

(3) Results and Discussion

The following table shows the results of determination of a constituentratio of genes in a system with no addition of a quenchingsubstance-labeled nucleic acid probe. The results show clearly that theamount of a change in fluorescence reduced with reducing amount of totalgenes because a ratio of fluorescent intensity (A and B) in the mimicsamples came close to a ratio of fluorescent intensity of thenon-hybridized probe. The determining values of the gene constituentratio in the mimic samples were consequently apart from the theoreticalvalue with the reducing amount of a total sum of genes. TABLE 7 Resultof quantitation of gene - constituent ratio (a system without additionof quenching substance - labeled probe) Quantitative Theoritical BODIPYTAMRA value value Relative Relative (target/ (target/ fluorescentfluorescent internal internal intensity intensity standard) standard)Non - 1.00 1.00 — — hybridizing probe 100% target 0.17 1.11 — — gene(BODIPY: a, TAMRA: b) 100% internal 0.96 0.14 — — standard gene (BODIPY:a′, TAMRA: b′) Mimic Sample 1 0.87 0.97 2.98 4.00 Total amount of gene:80 nM (BODIPY: A, TAMRA: B) Mimic Sample 2 0.94 0.98 2.27 Total amountof gene: 40 nM (BODIPY: A, TAMRA: B) Mimic Sample 3 0.98 0.99 1.39 Totalamount of gene: 20 nM (BODIPY: A, TAMRA: B)

The following table shows the results of determination of a constituentratio of genes in a system with addition of a quenchingsubstance-labeled nucleic acid probe. Even in this system, a ratio offluorescent intensity (A and B) in the mimic samples was reduced withreducing amount of total genes. Nevertheless, even in the case of theamount of a total sum of genes of the smallest 20 nM, the change in afluorescent intensity was observed to be about 1.5 to 2.0 times greateras compared with that for a non-hybridized probe. In the case of thereduced amount of a total sum of genes also, the obtained determiningvalues of gene constituent ratios were the ones about close to thetheoretical value. From the above results, it has become apparent that agene-constituent ratio can be accurately determined quantitatively byaddition of a quenching substance-labeled probe to reduce fluorescencefor the non-hybridized probe, and, as a result, a target gene can beaccurately determined quantitatively.

The quenching substance-labeled probe enabled to measure exactly thefluorescence derived from a probe hybridized to a target gene or aninternal standard gene by making the fluorescence for a non-hybridizingprobe quench. On the basis of this reason, the quenchingsubstance-labeled probe is in merit for all the target nucleic acidprobes (including an internal standard nucleic acid probe and adoubly-labeled probe) as described in the present application, and foran assaying method making use thereof.

In addition, in the case such that products amplified by a geneamplification method are targeted, the quenching substance-labeled probeis usable based on the same reason as described above. In such a case,it is necessary to conduct such a treatment as phosphorylation of the3′-end of a probe in the use of the quenching substance labeled probe,in which the 3′-end is not labeled, in order to inhibit to be used as aprimer (but, provided that the same shall not apply to the case when,subsequent to gene amplification, a constituent ratio of target genes isdetermined by adding a target nucleic acid probe (including an internalstandard nucleic acid probe and a doubly-labeled nucleic acid probe) anda quenching substance-labeled probe in the reaction mixture of the geneamplification). TABLE 8 Result of quantitation of gene - constituentratio (a system with addition of quenching substance - labeled probe)Quantitative Theoritical BODIPY TAMRA value value Relative Relative(target/ (target/ fluorescent fluorescent internal internal intensityintensity standard) standard) Non - 1.00 1.00 — — hybridizing probe 100%target 6.17 25.00 — — gene (BODIPY: a, TAMRA: b) 100% internal 33.333.75 — — standard gene (BODIPY: a′, TAMRA: b′) Mimic Sample 1 3.12 4.853.83 4.00 Total amount of gene: 80 nM (BODIPY: A, TAMRA: B) Mimic Sample2 2.04 3.03 4.36 Total amount of gene: 40 nM (BODIPY: A, TAMRA: B) MimicSample 3 1.56 1.99 3.64 Total amount of gene: 20 nM (BODIPY: A, TAMRA:B)

Example 10

Method for assaying a target nucleic acid using a novel mixturecomprising exonuclease according to the present invention

The example is below described, related to the additionally novel methodmaking use of both of a target nucleic acid probe and internal standardnucleic acid probe. This method makes use together of both of a targetnucleic acid probe and internal standard nucleic acid probe andadditionally an enzyme having exonuclease activity (hereinafter, calledsimply “exonuclease”). Its preferable example is described in FIG. 18.This method is premised on making use of 3′→5′exonuclease.

In this method, a target nucleic acid probe and an internal standardprobe, which are labeled doubly with a fluorescent dye and a quenchingsubstance are used. Fluorescent dyes to be used for labeling the targetnucleic acid probe and the internal standard probe are more preferableto be different each others. Further, on use of 3′→5′exonuclease,fluorescence-labeling portion of the probes are preferably 3′-endportions; quenching substance-labeling portion of the probes arepreferable to be different from the fluorescence-labeled portions. Thetarget nucleic acid probe is preferably a probe designed such that, onhybridization with one of the target gene and internal standard gene,the fluorescence-labeled 3′-end base of the probe is at leastnon-complementary to the one gene; and on hybridization with the othergene, the fluorescence-labeled 3′-end base of the probe is complementaryto the other gene. (In FIG. 18, the target nucleic acid probe isdesigned so that, on its hybridization to a target gene, thefluorescence-labeled 3′-end base of the probe is at leastnon-complementary to the target gene; and on hybridization with theinternal standard gene, the fluorescence-labeled 3′-end base of theprobe is complementary to the internal standard gene. On the other hand,the internal standard nucleic acid probe is designed so that, on itshybridization to an internal standard gene, the fluorescence-labeled3′-end base of the probe is at least non-complementary to the internalstandard gene; and on hybridization with the target gene, thefluorescence-labeled 3′-end base of the probe is complementary to thetarget gene.). In the target nucleic acid probe and internal standardnucleic acid probe, if the probes keep complete forms thereof, thefluorescence of the fluorescent dyes labeled at the probes is quenchedwith affection of the quenching substances labeled at a portion withinthe same molecules. However, if there is a mismatch at the 3′-end, the3′→5′exonuclease cleaves out the 3′-end base from the probes byrecognizing the mismatch on hybridization of the target nucleic acidprobe and internal standard nucleic acid probe with a target gene andinternal standard gene in the presence of the enzyme having acharacteristic such that the enzyme cleaves out the 3′-end base only inthe case of a mismatch of the 3′-end. Accordingly, each probe comes toemit specifically fluorescence on hybridization to any one of a targetgene and an internal standard gene (in FIG. 18, the target nucleic acidprobe is cleaved and emits fluorescence only on hybridization to atarget gene; the internal standard nucleic acid probe is cleaved andemits only on hybridization to an internal standard gene). A differencebetween the number of base pairs between the target nucleic acid probeand the target gene and the number of base pairs between the targetnucleic acid probe and the internal standard gene is only one base pair;that one base pair is the end base of the target nucleic acid; it can beunderstood that the target nucleic acid probe hybridizes to a targetgene and an internal standard gene with indiscriminating those (theinternal standard nucleic acid probe also is like this). Owing to this,a ratio between fluorescence-determining values for each probes canappropriately reflect a gene-constituent ratio between a target gene andan internal standard gene existing a system. Accordingly, it isconsidered that the amount of a target gene can be determinedquantitatively by this method also.

(1) Experimental Method

It was examined whether or not an existing ratio between a target geneand an internal standard gene existing in a system can be determined byusing the above described target nucleic acid probe, internal standardnucleic acid probe and 3′→5′exonuclease. In the experiment, a DNAsolution was prepared, wherein the solution comprised a target gene andinternal standard gene in various existing ratios between those; thetarget nucleic acid probe, internal standard nucleic acid probe and3′→5′exonuclease was added in the solution; subsequent to the addition,the solution was incubated for a determined time; and then afluorescence measurement was conducted. The fluorescence measurement wascarried out 95° C. A relation between a ratio of the amount offluorescence derived from each probes and a gene-constituent ratiobetween a target gene and an internal standard gene was examined; and itwas examined whether or not a gene-constituent ratio could be determinedbased on the obtained relative equation.

The following description shows detailed experimental conditions.

(2) Experiment Conditions

Target Gene and Internal Standard Gene

Target gene and internal standard gene: oligonucleotides were used.

Synthesis of oligonucleotide: by relying custom synthesis services(Espec oligoservices Inc.). Base sequence of target gene: 5′ TTGTC CGGAAAGGCC AGAGG AG-3′; Base sequence of internal standard gene: 5′-ATGTCCGGAA AGGCC AGAGG AG-3′;

Total final concentration as combined of standard gene and target gene:100 nM, 800 nM (target: internal standard=9:1, 3:1, 1:1, 1:3, 1:9).

Target Nucleic Acid Probe

Synthesis: by relying custom synthesis services (Espec oligoservicesInc.). Sequence: DABCYL-5′ CTCCT CTGGC CTTTC CGGAC AT 3′-BODIPY FL

Dye: BODIPY FL (like the dye used above), labeled portion: the 3′ end;

Quenching substance dye: DABCYL (like the dye used above), labeledportion: the 5′ end;

Final concentration: 200 nM.

Internal Standard Nucleic Acid Probe

Synthesis: by relying custom synthesis services (Espec oligoservicesInc.). Sequence: DABCYL-5′ CTCCT CTGGC CTTTC CGGAC AA3′-TAMRA

Dye: TAMRA (like the dye used above), labeled portion: the 3′ end;

Corrosive sublimate dye: DABCYL (like the dye used above), labeledportion: the 5′ end;

Final concentration: 200 nM.

-   -   3′→5′exonuclease: TaKaRa LA Taq (Takara Bio Inc.).    -   Buffer: A buffer attached with TaKaRa LA Taq was used.    -   Reaction time: 1 hr.

(3) Results and Discussion

FIG. 19 indicates the relation between a ratio offluorescence-determining values and a gene-constituent ratio of genes.Based on this figure, it is ascertained that there is a high co-relationbetween an amount ratio of changes in fluorescence and agene-constituent ratio regardless of the total amount of genes. Fromthese findings, it is clear that a gene-constituent ratio can bedetermined accurately, and, by utilizing this ratio, a target gene canbe determined quantitatively. Many heat-resistant enzymes for a PCRmethod are known to have a 3′→5′exonuclease activity. Because of thesefindings, the application of this method to a PCR method makes itunnecessary to add an enzyme having a 3′→5′exonuclease activity into anassaying system; this method could be carried out practically.

In this example, an instance utilizing a 3′→5′exonuclease activity hasbeen described; if there is a mismatching base pair, the use of anenzyme having a 5′→3′ exonuclease activity enables to determine a targetgene like use of the enzyme, 3′→5′exonuclease. In this case, afluorescence-labeling portion of a target nucleic acid probe andinternal standard nucleic acid probe should be preferably a 5′-end baseportion; a quenching substance-labeling portion is preferably a 3′-endbase portion.

Example 11

Calculating equation for making it possible to accurately determine aconcentration of target nucleic acid based on fluorescence measuringvalue (part 2).

The following description indicates an example making a doubly-labelednucleic acid probe and a quenching substance-labeled probe incombination, wherein the doubly-labeled nucleic acid probe has thecharacteristics such that a difference between the fluorescent intensityin regard to a dye labeled at one portion of a doubly-labeled nucleicacid probe on hybridization of a doubly-labeled nucleic acid probe witha target gene and the fluorescent intensity on the hybridization of theabove probe with an internal standard gene produces; and the fluorescentintensity in regard to a dye labeled at the other portion is in asimilar degree on hybridization with any of the target gene and theinternal standard gene. FIG. 20 illustrates a preferable example appliedwith this method. The one end of the doubly-labeled nucleic acid probeis labeled with a fluorescent dye (dye A) of such a type that thefluorescence for a fluorescent dye is capable of being quenched owing toan interaction of the dye with a G. Based on this labeling way, thefluorescence-quenching can occur because G is present in close proximityof the dye A on hybridization of the probe to a target gene; onhybridization to an internal standard gene, the fluorescence-quenchingcan not occur because G is absent in close proximity of the dye A. Theother end should be labeled with a dye B, although any limitation shouldnot be basically imposed on the dye, for which the fluorescent intensitybecomes to be in the same level on hybridization of the probe with atarget gene and on hybridization of the probe with an internal standardgene. Due to this labeling, the fluorescent intensity for dye B labeledon the other end portion of the probe is not affected by a sort of geneon hybridization of the probe with the gene to become like; thefluorescence for a non-hybridizing probe can be made to quench by aquenching substance-labeled probe; this make it possible to determinequantitatively the proportion of a hybridized probe. As a dye forlabeling the other end, a dye (dye B) which is difficult to be affectedby a base sequence is preferable. Further, a dye difficult to beaffected by a G is more preferable. In use of a dye for labeling aprobe, for which dye the fluorescent intensity is easy to be affected bya G, in order to enhance the intensity of fluorescence emitted from thedye, it is preferable to select a base sequence region where a G is lessin close proximity of the dye of the probe as a base sequence region ofa target gene to which the probe hybridize. On the basis of this reason,the base sequences of a target nucleic acid and an internal standardnucleic acid imposes in an instance some limitation on a design of aprobe. On the other hand, the use of a dye, for which the fluorescenceis not affected by a G, broaden a free choice in a probe design tobecome more preferable, because, if a G exists in close proximity of dyeB, the fluorescence for dye B is not affected by a G.

Illustrative are Cy dyes, Alexa dyes, Texas Red and the like as dyes forwhich fluorescent intensity is difficult to subject to the affection ofa base sequence,

(1) Experimental Method

In the experiment, the effect of the quenching substance-labeled nucleicacid probe was evaluated by comparison of determining values of existingratios of target genes in mimic samples. The determination of a existingratio of target genes was carried out by making use of the calculatingequation as described below.

Prior to the above experiment, a range of specific temperature was inadvance determined; in the range, the doubly-labeled nucleic acid probewas capable of hybridizing enough to only a target gene and an internalstandard gene, but of not hybridizing to the quenching substance-labelednucleic acid probe. The temperature range was 52 to 57° C. in theformer, and in the later not higher than 35° C. Based on the results, inascertaining experiment, the temperature was changed to 95° C., 52° C.,and 30° C. in this order and kept for one minute to an individualtemperature. The object of keeping the temperature to 52° C. was to makethe doubly-labeled nucleic acid probe hybridize to only a target geneand an internal standard gene. Subsequent to that, the keeping of 30° C.made the doubly-labeled nucleic acid hybridize to the quenchingsubstance-labeled nucleic acid probe to cause the fluorescence for thedoubly-labeled nucleic acid to be quenched.

<Calculating Equation>

This example illustrates a calculating method for determining agene-constituent ratio quantitatively.

Signs usable in the calculating equation are below defined. A dye isdefined as dye A, which dye causes a difference between the fluorescentintensity for the doubly-labeled probe on its hybridization to a targetgene and the fluorescent intensity for the doubly-labeled probe on itshybridization to an internal standard gene; a dye is defined as dye B,which dye causes no difference.

y: a proportion of a hybridized probe;

1−y: a proportion of a non-hybridized probe;

x: a proportion of a target gene;

1−x: a proportion of an internal standard gene;

A: a ratio of the fluorescent intensity for dye A of the doubly-labelednucleic acid probe on its 100%-hybridization with the quenchingsubstance-labeled probe in use of a practical sample to the fluorescentintensity of dye A of the doubly-labeled nucleic probe on its100%-hybridization with a quenching substance-labeled probe;

a: a ratio of the fluorescent intensity of dye A of the doubly-labelednucleic acid probe on its 100%-hybridization with a target nucleic acidto the fluorescent intensity of dye A of the doubly-labeled nucleic acidprobe on its 100%-hybridization with a quenching substance-labeledprobe;

a′: a ratio of the fluorescent intensity of dye A of the doubly-labelednucleic acid probe on its 100%-hybridization with an internal standardnucleic acid to the fluorescent intensity of dye A of the doubly-labelednucleic acid probe on its hybridization with a quenchingsubstance-labeled probe;

B: a ratio of the fluorescent intensity of dye B of the doubly-labelednucleic acid probe on its hybridization in use of a practical sample tothe fluorescent intensity of dye B of the doubly-labeled nucleic probeon its 100%-hybridization with a quenching substance-labeled probe;

b: a ratio of the fluorescent intensity of dye B of the doubly-labelednucleic acid probe on its 100%-hybridization with a target gene and aninternal standard gene to the fluorescent intensity of dye B of thedoubly-labeled nucleic acid probe on its 100%-hybridization with aquenching substance-labeled probe;

The fluorescent intensity proportion (A) of dye A in use of a practicalsample is capable of being represented as follows:A=(1−y)+axy+(1−x)a′y=1−y+axy+a′y−a′xy  (1)

If the equation (a) is deformed, a proportion (x) of a target gene iscapable of being represented as follows:x=(1−y−A+a′y)/(a′y−ay)  (2)

On the other hand, a fluorescent intensity (B) for dye B in use of apractical sample is capable of being represented.B=by +1−y  (3)

If the equation (3) is deformed, a proportion (y) of a hybridized probeis capable of being represented as follows:Y=(B−1)/(b−1)  (4)

The equation (2) is incorporated with the equation (4); the resultantequation is represented as follows:x=(1−(B−1)/(b−1)−A+a′(B−1)/(b−1))/(a′(B−1)/(b−1)−a(B−1)/(b−1))=(b−B−Ab+A+a′B−a′)/(a′B−a′−aB+a)  (5)

The items other than x are determined experimentally; a proportion of atarget gene is thought to be capable of being calculated by an equation(5).

(2) Experimental Conditions

Target Gene and Internal Standard Gene

The same as in Example was used.

Total final concentration as combined of internal standard gene andtarget gene in a mimic sample: 20 nM, 40 nM, 80 nM (target:internalstandard=4:1)

Base Sequence and Structure of Doubly-Labeled Nucleic Acid Probe BODIPYFL-5′ ACTAC TGACT GACTG ACTGA CTGCT CC 3′- Cy5

Final concentration: 400 nM

Incidentally, those were like Example 6 except the 3′-end is labeledwith Cy5.

Quenching Substance-Labeled Probe:

DABCYL (Glean Research Inc., US) was used as a quenching substance. Basesequences are described as follows:

Quenching substance-labeled probe A: 5′ GTCAG TCAGT AGTG3′-DABCYL

The 3′-end was labeled with DABCYL.

Quenching substance-labeled probe B: DABCYL-5′ GGGAG CAGTC AGTCA

The 5′-end was labeled with DABCYL.

(The underlined portions show the hybridization region of thedoubly-labeled probe)

Synthesis of above oligonucleotides: by relying custom synthesisservices (Espec oligoservices Inc.).

Final concentration of quenching substance-labeled probe: 800 nM

Fluorescence for Cy5 was measured by an exciting wavelength: 600 nm andmeasuring wavelength: 670 nm.

Other conditions (used buffer, used apparatus, used instrument, and thelike) were like those of Example 9.

(3) Results and Discussion

The below table shows the results of quantitation of a constituent ratioof genes. In this system also, a ratio of fluorescent intensity (A andB) for BODIPY FL in the mimic samples was reduced with reducing amountof total genes. However, in the case of the amount of a total sum ofgenes of the smallest 20 nM also, the determining values of the geneconstituent ratio in the mimic samples were almost close to thetheoretical value. From the above results, it has become apparent that agene-constituent ratio can be accurately determined quantitatively byaddition of a quenching substance-labeled probe to reduce fluorescencefor the non-hybridized probe, and, as a result, a target gene can beaccurately determined quantitatively. In addition, the calculationmethod as described in this Example also was apparently useful fordetermining a gene-constituent ratio quantitatively.

Likewise in Example 9, the quenching substance-labeled probe makesfluorescence for the non-hybridized probe reduce; thereby, the probemakes it possible to determine accurately fluorescence derived from thehybridization of the probe with a target gene and an internal standardgene. On the basis of the above reason, the quenching substance-labeledprobe is advantageous for all target nucleic acid probes (including aninternal standard nucleic acid probe and a doubly-labeled nucleic acidprobe) and all assaying methods making use thereof as described in thisapplication.

Additionally, in the case of targeted amplification products amplifiedby any gene-amplification method also, the quenching substance-labeledprobe is profitable based on the same reason as the above one. TABLE 9Result of quantitation of gene - constituent ratio QuantitativeTheoritical BODIPY Cy5 value value Relative Relative (target/ (target/fluorescent fluorescent internal internal intensity intensity standard)standard) Non - 1.00 1.00 — — hybridizing probe 100% target 2.33 12.50 —— gene (BODIPY: a, Cy5: b) 100% internal 11.11 — — standard gene(BODIPY: a′, Cy5: b) Mimic Sample 1 1.59 3.23 4.22 4.00 Total amount ofgene: 80 nM (BODIPY: A, Cy5: B) Mimic Sample 2 1.31 2.13 3.84 Totalamount of gene: 40 nM (BODIPY: A, Cy5: B) Mimic Sample 3 1.14 1.51 3.68Total amount of gene: 20 nM (BODIPY: A, Cy5: B)

INDUSTRIAL APPLICABILITY

A novel mixture according to the present invention is capable of providea method for assaying a nucleic acid, the method producing advantageouseffects as described in the above description. The present invention,therefore, can greatly contribute to genetic engineering, medicals,medical arts, agricultural techniques, development of various biologicaltechniques, development of complex microbes usable in wastes-treatmentplants and the like.

1. A novel mixture or novel reaction solution for assaying one or two ormore target nucleic acids, which comprises one or two or more belownucleic acid probes for a homogenous solution system and one or two ormore below internal standard nucleic acids, or further one or two ormore below internal standard nucleic acid probes: A) said nucleic acidprobe for a homogenous solution system (hereinafter, called a “targetnucleic acid probe”) having below characteristics a) said target nucleicacid probe for a homogenous solution system is formed of one singlestranded oligonucleotide; b) said target nucleic acid probe for ahomogenous solution system is labeled with one or two or more moleculeof fluorescent dyes of one or two or more kinds at least one of both endportions and/or at least one of base moieties in the chain, at least oneof sugar moieties and/or at least one of phosphate moietes of theoligonucleotide; c) said target nucleic acid probe for a homogenoussolution system enables a fluorescent character to change on hybridizingwith a target nucleic acid and/or an internal standard nucleic acid; d)said target nucleic acid for a homogenous solution system is capable ofhybridizing without discriminating with a target nucleic acid or aninternal standard nucleic acid; e) said target nucleic acid probe for ahomogenous solution system is capable of producing a difference betweenthe amount of change in a fluorescent character on hybridization with aninternal standard nucleic acid and the amount of change in a fluorescentcharacter on hybridization with a target nucleic acid. B) Said internalstandard nucleic acid: Said internal standard nucleic acid has astructure different in at least a portion from the structure of a regionof a target nucleic acid corresponding to the target nucleic acid probe,and said internal standard nucleic acid is capable of causing adifference between the amount of a change in a fluorescent character onhybridizing with the target nucleic acid probe and the amount of achange in a fluorescent character on the hybridization of a targetnucleic acid with the target nucleic acid probe. C) Said internalstandard nucleic acid probe: Said internal standard nucleic acid probehas the following characteristics: Said internal standard nucleic acidprobe has said characteristics a) to e) of the target nucleic acidprobe, wherein a fluorescent labeling portion and a fluorescentcharacter of a labeled fluorescent dye each are different from that ofthe target nucleic acid probe.
 2. A novel method for assaying a targetnucleic acid, comprising assaying one or two or more target nucleicacids using the novel mixture according to claim
 1. 3. A novel methodfor assaying a target nucleic acid according to claim 2, wherein saidnovel mixture comprises the below target nucleic acid probe and apredetermined concentration of the internal standard nucleic acidaccording to claim 1: said target nucleic acid probe (a recognizablenucleic acid probe), wherein said target nucleic acid probe according toclaim 1 is not complementary to either a target nucleic acid or aninternal standard nucleic acid at least one or two or morefluorescent-labeled portions.
 4. A novel method for assaying a targetnucleic acid, in which said method comprises quenching an fluorescentemission of the target nucleic acid probe or the internal standardnucleic acid probe not hybridized with either an internal standardnucleic acid or a target nucleic acid in the novel method for assaying atarget nucleic acid according to claim 2 or
 3. 5. A novel method forassaying a target nucleic acid, in which comprises: multiplying one orplural target nucleic acids and internal standard nucleic acids in areaction system comprising one or plural internal standard nucleic acidsaccording to claim 1 until an optional phase of from a beginning phaseto a stationary phase by a gene amplification method; and determiningstarting concentrations of one or plural target nucleic acids prior tothe amplification using the resultant reaction solution or the amplifiedproduct as a sample.
 6. A novel mixture for assaying one or pluraltarget nucleic acids based on measurement of a Tm value, in which saidmixture comprises a pair of the below nucleic acid and the belowinternal standard nucleic acid: Said nucleic acid probe: Said nucleicacid probe is a single stranded oligonucleotide labeled with one or twoor more fluorescent dyes, wherein said nucleic acid probe is capable ofhybridizing with a target nucleic acid and the below internal standardnucleic acid, and causing changes in fluorescent characters of thefluorescent dyes labeled thereon on hybridization with the targetnucleic acid and internal standard nucleic acid, wherein, in the casewhen said nucleic acid probe is plural, the fluorescent dyes labeled onsaid plural nucleic acid probes each are different. Said internalstandard nucleic acid: The base sequence of a portion of said internalstandard nucleic acid hybridizing with said nucleic acid probe isdifferent in part from the base sequence of a portion of a nucleic acidhybridizing with said nucleic acid probe.
 7. A novel method for assayinga target nucleic acid, in which said novel method comprises measuringfluorescent intensity using said novel mixture according to claim 6 withchanging temperature under the presence of plural target nucleic acids;and determining a target nucleic acid by the following procedures: Saidprocedures comprising: 1) drawing a curve dependent to changedfluorescent intensity measured; 2) differentiating the resulting curve;3) integrating the resulting peak(s) and determining the area(s) of thepeak(s); 4) calculating a ratio(s) of the resulting peak area(s) of theinternal standard nucleic acid and the resulting peak area(s) of thetarget nucleic acid; 5) multiplying the concentration of the internalstandard nucleic acid by said ratio.
 8. A calculating equationrepresented by the following equation for calculating accurately atarget nucleic acid based on measuring values of a change in an opticalcharacter in said novel method for assaying a target nucleic acidaccording to claims 2 and 3:x=(−a′−B+Ba′+b′+A−Ab′)/(b′−b−Ab′+Ab−a′+a+Ba′−Ba) wherein said equationis under the below conditions; and said signs have the below meanings:Said conditions are as follows: said novel method uses thedoubly-labeled nucleic acid probe, wherein said nucleic acid probe islabeled with dyes A and B. Said signs are as follows: x: a proportion ofa target gene; A: a ratio of the fluorescent intensity of dye A of thedoubly-labeled nucleic acid probe on no hybridization in use of apractical sample to the fluorescent intensity of dye A of thedoubly-labeled nucleic probe on no hybridization; a: a ratio of thefluorescent intensity of dye A of the doubly-labeled nucleic acid probeon its 100%-hybridization with a target nucleic acid to the fluorescentintensity of dye A of the doubly-labeled nucleic acid probe on nohybridization; a′: a ratio of the fluorescent intensity of dye A of thedoubly-labeled nucleic acid probe on its 100%-hybridization with aninternal standard nucleic acid to the fluorescent intensity of dye A ofthe doubly-labeled nucleic acid probe on no hybridization; B: a ratio ofthe fluorescent intensity of dye B of the doubly-labeled nucleic acidprobe on hybridization using a practical sample to the fluorescentintensity of dye B of the doubly-labeled nucleic probe on nohybridization; b: a ratio of the fluorescent intensity of dye B of thedoubly-labeled nucleic acid probe on its 100%-hybridization with atarget gene to the fluorescent intensity of dye B of the doubly-labelednucleic acid probe on no hybridization; b′: a ratio of the fluorescentintensity of dye B of the doubly-labeled nucleic acid probe on its100%-hybridization with an internal standard nucleic acid to thefluorescent intensity of dye B of the doubly-labeled nucleic acid probeon no hybridization.
 9. A calculating equation represented by thefollowing equation for calculating accurately a target nucleic acidbased on measuring values of a change in an optical character in saidnovel method for assaying a target nucleic acid according to claims 2and 3:x=(b−B−Ab+A+a′B−a′)/(a′B−a′−aB+a) wherein said equation is under thebelow conditions; and said signs have the below meanings: Saidconditions are as follows: said novel method uses said doubly-labelednucleic acid probe, wherein said nucleic acid probe is labeled with dyesA and B. Said signs are as follows: x: a proportion of a target gene; A:a ratio of the fluorescent intensity of dye A of the doubly-labelednucleic acid probe on its 100%-hybridization with a quenchingsubstance-labeled nucleic acid probe in the case making use of apractical sample to the fluorescent intensity of dye A of thedoubly-labeled nucleic acid probe on its 100%-hybridization with aquenching substance-labeled nucleic acid probe a: a ratio of thefluorescent intensity of dye A of the doubly-labeled nucleic acid probeon its 100%-hybridization with a target nucleic acid to the fluorescentintensity of dye A of the doubly-labeled nucleic acid probe on its100%-hybridization with a quenching substance-labeled nucleic acidprobe; a′: a ratio of the fluorescent intensity of dye A of thedoubly-labeled nucleic acid probe on its 100%-hybridization with aninternal standard gene to the fluorescent intensity of dye A of thedoubly-labeled nucleic acid probe on its 100%-hybridization with aquenching substance-labeled nucleic acid probe; B: a ratio of thefluorescent intensity of dye B of the doubly-labeled nucleic acid probeon its 100%-hybridization with a quenching substance-labeled nucleicacid probe in the case making use of a practical sample to thefluorescent intensity of dye B of the doubly-labeled nucleic probe onits 100%-hybridization with a quenching substance-labeled nucleic acidprobe; b: a ratio of the fluorescent intensity of dye B of thedoubly-labeled nucleic acid probe on its 100%-hybridization with atarget gene and an internal standard gene to the fluorescent intensityof dye B of the doubly-labeled nucleic acid probe on its100%-hybridization with a quenching substance-labeled nucleic acidprobe.
 10. A kit for assaying a nucleic acid, in which said kit comprisethe novel mixture according to claim 1 or
 6. 11. A target nucleic acidprobe or a doubly-labeled nucleic acid probe, wherein said targetnucleic acid probe or doubly-labeled nucleic acid probe is describedabove and has at least any one of the below structures:
 1. Saidstructures of said target nucleic acid probe, wherein 1) said structurehas a portion not complementary to a target nucleic acid and/or aninternal standard nucleic acid at a end portion or both end portions; 2)in the above 1), said nucleic acid probe is labeled with a fluorescentdye at one end portion not complementary to the target nucleic acidand/or an internal standard nucleic acid, having a cytosine (a C) or aguanine (a G) in a range of the 1st base to 3rd base from the labeledbase in a fluorescent dye-labeled portion (the labeled base is countedas 1st base); 3) in the above 1), the other one end portion notcomplementary to a target nucleic acid and/or an internal standardnucleic acid is at a portion opposite said one end portion labeled witha fluorescent dye; 4) in the above 1), the other end portion notcomplementary to a target nucleic acid and/or an internal standardnucleic acid is in a range of one to four bases in a chain length; 5) inthe above 1), if any of two bases of a target nucleic acid correspondingto the both ends of the target nucleic acid probes is a G, that of theinternal standard nucleic acid is a base other than a G; if that of thetarget nucleic acid is a base other than a G, that of the internalstandard nucleic acid is a G.
 2. Structures of said doubly-labeledtarget nucleic acid probe, wherein 6) portions labeled with fluorescentdyes were at least two bases; 7) the bases according to the above 6) aretwo C's; 8) the two C's according to the above 7) are the bases of bothends; 9) the base sequence according to the above 6) are complementaryto a target nucleic acid or an internal standard nucleic acid excludingboth end portion (at least from one base to three bases in chain lengthfrom an end base, the end base counted as the 1st base); 10) thedoubly-labeled nucleic acid probe according to the above 6) or 9) is adoubly-labeled nucleic acid probe making at one portion a differencebetween the amount of a change in a fluorescent character onhybridization with a target nucleic acid and that on hybridization withan internal standard nucleic acid, but not making such a difference atthe other portion;
 3. Structures common to said target nucleic acidprobe and said doubly-labeled nucleic acid probe: wherein 11) in any oneof the above 1) to 10), a base sequence of said target nucleic acidprobe or a doubly-labeled nucleic acid is at least complementary to atarget nucleic acid or an internal standard nucleic acid excluding bothend base portions (a base sequence of at least one base to three basesin length; the end base counted as the 1st base); 12) in any one of theabove 1) to 11), said target nucleic acid probe or said doubly-labelednucleic acid probe has a base sequence completely complementary to atarget nucleic acid and an internal standard nucleic acid; 13) in anyone of the above 1) to 12), if the base of a target nucleic acidcorresponding to an end base of a target nucleic acid probe or adoubly-labeled nucleic acid probe is taken as the 1st base, the numberof a G of the corresponding target nucleic acid or internal standardnucleic acid in a range of from the 1st base to the 3rd base is largerin a target nucleic acid than in an internal standard nucleic acid, orsmaller in a target nucleic acid than in an internal standard nucleicacid; 14) in any one of the above 1) to 13), if the base of a targetnucleic acid corresponding to both end base of a target nucleic acidprobe or a doubly-labeled nucleic acid probe is taken as the 1st base,the number of a G of the corresponding target nucleic acid and internalstandard nucleic acid in a range of from the 1st base to the 3rd base islarger in a target nucleic acid than in an internal standard nucleicacid in one end region, and in the other end region smaller in a targetnucleic acid than or equal in an internal standard nucleic acid in theother end rejoin; or in one end region smaller in a target nucleic acidthan in an internal standard nucleic acid and in the other end regionlarger in a target nucleic acid than or equal in an internal standardnucleic acid; 15) a novel mixture according to claim 1 or 5, wherein, inany one of the above 1 to 14, the base of a target nucleic acidcorresponding to one end base of the target nucleic acid probe or thedoubly-labeled nucleic acid probe is a base other than a G and the basecorresponding to the other end base is a G. 16) in any one of theabove 1) to 15), if any of two bases of a target nucleic acidcorresponding to both end bases a target nucleic acid probe is a G, thatof an internal standard nucleic acid is a base other than a G; and ifthat of a target nucleic acid is a base other than a G, that of aninternal standard nucleic acid is a G; 17) in anyone of the above, ifthe base of a target nucleic acid corresponding to both end base of atarget nucleic acid probe or a doubly-labeled nucleic acid probe istaken as the 1st base, the corresponding base sequence of a targetnucleic acid and internal standard nucleic acid is different in one endregion in a range of from the 1st base to the 3rd base, but the same inthe other end region; 18) in anyone of the above, if the base of atarget nucleic acid corresponding to both end base of a target nucleicacid probe or a doubly-labeled nucleic acid probe is taken as the 1stbase, the corresponding base sequence of a target nucleic acid andinternal standard nucleic acid in a range of from the 1st base to the3rd base is different in both end region.